Jun 26, 2026

Processing human brain tissue and cultured cells for population-scale SQK-PCS114 Oxford Nanopore long-read cDNA-PCR sequencing SOP

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwbw22vmk/v1
  • Jackson Mingle1,
  • Cedric Kouam1,
  • Pilar Álvarez Jerez2,
  • Allison Evans1,
  • Abraham Moller3,
  • Breeana Baker1,
  • Cory Weller3,
  • Kimberly Paquette1,
  • Janet Brooks4,
  • Spencer M Grant5,
  • Melissa Meredith3,
  • Joanna Palade1,
  • Laksh Malik1,
  • Yilei Fu6,
  • Xinchang Zheng6,
  • Fritz J Sedlazeck7,
  • Luigi Ferrucci8,
  • Xylena Reed1,
  • Mike Nalls3,
  • Mark Cookson9,
  • Kendall Van Keuren-Jensen10,
  • Elizabeth Hutchins11,
  • Miten Jain12,
  • Kimberley J Billingsley1
  • 1Center for Alzheimer's and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA;
  • 2Center for Alzheimer's and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, University College London, London, UK;
  • 3Center for Alzheimer's and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA, DataTecnica, Washington, DC, USA;
  • 4National Institute on Aging, National Institutes of Health, Bethesda, MD, USA;
  • 5Center for Alzheimer's and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA, Department of Clinical Neurosciences, School of Clinical Medicine, The University of Cambridge, Cambridge, UK;
  • 6Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA;
  • 7Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, Department of Computer Science, Rice University, Houston, TX, USA;
  • 8Translational Gerontology Branch, National Institute on Aging (NIA/NIH), Baltimore, MD, USA;
  • 9Center for Alzheimer's and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA, Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA;
  • 10Bioinnovation and Genome Sciences Division, Translational Genomics Research Institute, Phoenix, AZ, USA;
  • 11Center for Alzheimer's and Related Dementias, National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA, DataTecnica, Washington, DC, USA, Bioinnovation and Genome Sciences Division, Translational Genomics Research Institute, Phoenix, AZ, USA;
  • 12Department of Bioengineering, Department of Physics, Khoury College of Computer Sciences, Northeastern University, Boston, MA, USA
  • NIH Center for Alzheimer's and Related Dementias
Jackson Mingle
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwbw22vmk/v1
Protocol Citation: Jackson Mingle, Cedric Kouam, Pilar Álvarez Jerez, Allison Evans, Abraham Moller, Breeana Baker, Cory Weller, Kimberly Paquette, Janet Brooks, Spencer M Grant, Melissa Meredith, Joanna Palade, Laksh Malik, Yilei Fu, Xinchang Zheng, Fritz J Sedlazeck, Luigi Ferrucci, Xylena Reed, Mike Nalls, Mark Cookson, Kendall Van Keuren-Jensen, Elizabeth Hutchins, Miten Jain, Kimberley J Billingsley 2026. Processing human brain tissue and cultured cells for population-scale SQK-PCS114 Oxford Nanopore long-read cDNA-PCR sequencing SOP . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwbw22vmk/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
This method is in development. ONT has published updated PCR guidelines that will be implemented for future cohorts. The updated PCR parameters can be found on the ONT PCS114 website, and are as follows: Incubate in a thermoycler using the following protocol: 98 °C for 00:00:10 (5x) - Initial denaturation 50 °C for 00:00:30 (5x) - Initial annealing 68 °C for 00:03:00 (5x) - Initial extension 98 °C for 00:00:10 (17x) - Denaturation 68 °C for 00:03:00 (17x) - Annealing and extension Hold at 4 °C Lid temperat
Created: March 01, 2025
Last Modified: June 27, 2026
Protocol  Integer ID: 123614
Keywords: Long-read sequencing, Oxford Nanopore sequencing, Human tissue disruption, RNA extraction, Brain tissue extraction, PCS114 Oxford Nanopore, Transcriptome sequencing, Transcriptome, Sequencing, Sequencing SOP, Human brain tissue, RNA, Dementia, Alzheimer's disease, sequencing data, read rna, pcs114 oxford nanopore, nih center for alzheimer, sequencing sop, processing human brain tissue, human brain tissue, patients with alzheimer, read cdna, lewy body dementia, alzheimer, related dementia, data from patient
Abstract
At the NIH Center for Alzheimer's and Related Dementias (CARD) (https://card.nih.gov/research-programs/long-read-sequencing), we will generate long-read sequencing data from patients with Alzheimer's disease, frontotemporal dementia, and Lewy body dementia, as well as healthy subjects. With this research, we will build a public resource consisting of long-read transcriptome sequencing data from a large number of confirmed patients with Alzheimer's disease and related dementias and healthy individuals. To generate this large-scale nanopore sequencing data, we have developed a protocol for processing and long-read RNA sequencing of human brain tissue.


Figure 1. Overview of the RNA extraction and ONT sequencing protocol

Materials
Materials:
AB
MaterialVendor (Part Number)
Sterile weigh boatsAny major lab supplier (MLS)
Single edge razor bladesAny MLS
Laboratory spatulasAny MLS
TweezersAny MLS
Plastic wrapAny MLS
2 mL Protein LoBind tubesEppendorf (022431102)
Ethanol (96-100%)Any MLS
RNaseZapThermo Fisher Scientific (AM9780)
RNeasy Lipid Tissue Mini kitQiagen (74804)
Stainless Steel Beads, 5 mmQiagen (69989)
ChloroformSigma-Aldrich (C2432)
0.5 M EDTAThermo Fisher Scientific (J15694.AP)
Trisodium citrate dihydrateMillipore Sigma (S1804)
Ammonium sulfateMillipore Sigma (A4418)
Hydrochloric acidMillipore Sigma (H9892)
0.2µm PES sterile disposable filter unitThermo Fisher Scientific (567-0020)
1.5 mL DNA LoBind tubesEppendorf (022431021)
Qubit RNA BR assay kitThermo Fisher Scientific (Q10211)
Qubit Flex Assay Tube StripsThermo Fisher Scientific (Q33252)
RNA ScreenTapeAgilent Technologies, Inc. (5067-5576)
RNA ScreenTape LadderAgilent Technologies, Inc. (5067-5578)
RNA ScreenTape Sample BufferAgilent Technologies, Inc. (5067-5577)
TapeStation tubesAgilent Technologies, Inc. (401428)
TapeStation capsAgilent Technologies, Inc. (401425)
2-MercaptoethanolSigma-Aldrich (63689)
RNeasy Plus Mini kitQiagen (74134)
QIAshredderQiagen (79654)
cDNA-PCR sequencing kitOxford Nanopore Technologies (SQK-PCS114)
0.2 mL thin-walled PCR tubesThermo Fisher Scientific (AB-0620B)
0.2 mL 8-tube stripsUSA Scientific (1402-4708)
Nuclease-free waterAny MLS
SIRV-Set 4Lexogen (M14103-8-0100)
T3 DNA LigaseNEB (M0317L)
RNaseOUTThermo Fisher Scientific (10777019)
Lambda ExonucleaseNEB (M0262L)
Uracil-Specific Excision Reagent (USER) EnzymeNEB (M5505L)
RNAClean XP beadsBeckman Coulter (A63987)
10 mM dNTP SolutionNEB (N0447S)
Maxima H Minus Reverse TranscriptaseThermo Fisher Scientific (EP0753)
Ex Premier DNA PolymeraseTakara Bio (RR370A)
Thermolabile Exonuclease INEB (M0568L)
AMPure XP beadsBeckman Coulter (A63882)
Qubit 1X dsDNA HS assay kitThermo Fisher Scientific (Q33231)
TapeStation D5000 ReagentsAgilent Technologies, Inc. (5067-5589)
TapeStation D5000 ScreenTapeAgilent Technologies, Inc. (5067-5588)
PromethION flow cellOxford Nanopore Technologies (FLO-PRO114M)
Table 1. Required materials
Equipment:
AB
EquipmentVendor (Part Number)
1.5/2 mL microcentrifuge tube cooling blockAny major lab supplier (MLS)
Analytical balanceAny MLS
BeakerAny MLS
Graduated cylinderAny MLS
Magnetic stirring rodAny MLS
Magnetic stir plateAny MLS
pH meterAny MLS
TissueLyser Single-Bead Dispenser, 5 mmQiagen (69965)
TissueLyser IIIQiagen (9003240)
TissueLyser Adapter Set 2 x 24Qiagen (69982)
DynaMag-2 MagnetThermo Fisher Scientific (12321D)
MicrocentrifugeEppendorf (5404000413)
Qubit Flex fluorometerThermo Fisher Scientific (Q33327)
Fluoroskan microplate fluorometerThermo Fisher Scientific (5200110)
4200 TapeStation systemAgilent Technologies Inc. (G2991B)
ThermoMixerEppendorf (5382000023)
Vortex mixerAny MLS
MinicentrifugeAny MLS
Thermal cyclerBio-Rad (1851197)
PromethION 24 or 48 sequencing unitOxford Nanopore Technologies (PRO-SEQ024 or PRO-SEQ-048)
Picus 2 Electronic Single Channel PipetteSartorius (LH-747081)
Table 2. Required equipment
Materials for NGS STAR:
AB
MaterialVendor (Part Number)
0.5 mL tubesSarstedt (72.730.305)
2 mL tubesSarstedt (72.693.005)
PCR ComfortLidHamilton (814300)
MIDI plateThermo Fisher Scientific (AB-0859)
HSP plateBio-Rad (HSP9601)
50 µL tipsHamilton (235979)
300 µL tipsHamilton (235903)
1000 µL tipsHamilton (235940)
20 mL reservoirHamilton (10161052)
60 mL reservoirHamilton (56694-01)
300 mL reservoirHamilton (56669-01)
96-well plate for TapeStationAgilent (5042-8502)
Table 3. Additional materials required for automated library preparation on Hamilton NGS STAR

Equipment for NGS STAR:
AB
EquipmentVendor (Part Number)
NGS STARHamilton (STAR)
Magnetic Stand-96Thermo Fisher Scientific (AM10027)
Table 4. Additional equipment reqired for automated library preparation on Hamilton NGS STAR
Part 1a: Extracting RNA from frozen human brain tissue using Qiagen RNeasy Lipid Tissue Mini kit
Place the following supplies on dry ice and allow to chill for 00:10:00 prior to use:
  • Sterile weigh boats
  • Razor blades
  • Spatulas
  • Tweezers
  • Empty 2 mL Eppendorf Protein LoBind tubes - labeled with sample ID
  • Cooling block

Note: Reusable metal tools (spatulas, tweezers) must be cleaned with 70% ethanol, RNaseZap, and distilled water prior to use. Apply a layer of plastic wrap to the dry ice and place metal tools on the plastic wrap to chill them prior to use.
If necessary, prepare RNALater solution as follows:
  • Prepare 1 M trisodium citrate dihydrate solution by adding 147 g trisodium citrate dihydrate to 400 mL nuclease-free water in a clean beaker, stirring until dissolved, then adding nuclease-free water up to 500 mL. Filter the solution with a 0.2 µM filter bottle and store at room temperature.
  • Add approximately 800 mL nuclease-free water to a clean beaker.
  • Add a stirring rod and place on hot plate stirrer on low heat, adjusting the speed to ensure adequate mixing.
  • Add 40 mL 0.5 M EDTA to beaker.
  • Add 25 mL 1 M trisodium citrate dihydrate to beaker.
  • Using an analytical balance, measure 700 g ammonium sulfate and add to beaker.
  • Stir until fully dissolved, then allow to cool.
  • Adjust to pH 5.2 with hydrochloric acid.
  • Pour contents of beaker into graduated cylinder.
  • Top up graduated cylinder with nuclease-free water until it reaches 1000 mL.
  • Pour contents of graduated cylinder into 0.2 µM filter bottle attached to vacuum.
  • Turn on vacuum to filter solution into bottle.
  • Turn off vacuum once complete, unscrew filter top, then quickly cap the bottle.
  • Label with name, date, and "RNALater".
  • Store at room temperature.
  • Prior to incubation with brain tissue, store RNALater solution on ice.
Obtain tissue samples from -80°C freezer and place them on dry ice.
Don all necessary protective equipment prior to setting up the biosafety cabinet:
  • Disposable lab coat
  • Gloves (2 pairs)
  • Face shield or mask
Wipe down biosafety cabinet with 70% ethanol, RNaseZap, and distilled water. Ensure scale is set up properly and level. Place dry ice buckets with supplies and samples in biosafety cabinet.
Remove cooling block from dry ice. Place sterile weigh boat onto cooling block.
Weigh labeled empty 2 mL Eppendorf Protein LoBind tube and tare the scale, ensuring the tube is centered on the scale.

Note: If there's ice surrounding the tube, wipe it off before taring. When weighing the sample, work quickly. As the dry ice surrounding the tube evaporates, the weight may fluctuate.
Pour frozen brain tissue onto sterile weigh boat on cooling block. With the razor blade in one hand, and with the other hand shielding the weigh boat to prevent frozen tissue from flying onto the surface, cut the brain tissue firmly.
Pick up the cut piece of brain tissue with the razor blade and transfer to the empty labeled tube. Weigh the tube immediately and record the weight. If necessary, continue to cut the brain tissue to reach the target weight. Return to dry ice immediately.

Note: 20-30 mg is required for RNA extraction for long-read sequencing. Input requirements will vary per brain region.

Note: It is crucial to prevent brain tissue from thawing during the cutting process. Once the brain tissue is removed from dry ice, cut and weigh the tissue quickly and return to dry ice immediately. Thawing of brain tissue during cutting could lead to RNA extractions with reduced quality.

Note: For flaky or powdery samples, clean metal tweezers or spatulas can be used to transfer tissue to the tube.
Return any unused brain tissue on the weigh boat to a tube for permanent storage. Return to dry ice immediately.
Dispose of the razor blade in the sharps container. Dispose of the weigh boat, top layer of gloves, and empty tube formerly containing brain tissue (if applicable) in Medical Pathological Waste (MPW) box. Return the cooling block to dry ice.
Add 1 mL RNALater solution directly to sample tubes. Ensure all tissue is fully submerged in solution. Store on wet ice during processing.
Repeat steps 5-12 for additional brain tissue samples. Wipe down biosafety cabinet with 70% ethanol, RNaseZap, and distilled water, and put on a new top layer of gloves between each sample to prevent cross-contamination. Keep all samples on dry ice when not in use.
When finished, wipe down biosafety cabinet with RNaseZap, 70% ethanol, and distilled water. Clean reusable metal tools with 10% bleach and water, or RNaseZap, 70% ethanol, and distilled water.
Incubate brain tissue samples in RNALater in 4°C refrigerator overnight.
Complete the following preparation steps:
  • Place TissueLyser adapters in -20°C freezer
  • Chill centrifuge to 4 °C
  • Wipe down all surfaces, pipettes, and magnetic racks with RNaseZap and 70% ethanol
Obtain tissue samples and place in pre-chilled cooling block.

Note: Until noted later in the protocol, the following steps should be performed in a fume hood to protect from exposure to QIAzol and chloroform.
Remove RNALater solution from tube and discard in designated RNALater waste bottle.
Using the Qiagen bead dispenser, add one 5mm stainless steel bead to each sample.

Note: To prevent brain tissue from flying out of the tube, dispense bead into tube cap, then close the tube.
Add 1 mL QIAzol to each sample. Ensure all brain tissue chunks are submerged in QIAzol using the pipette tip.
Remove the TissueLyser adapters from -20°C freezer. Load samples into the middle rows of the TissueLyser adapters. Ensure TissueLyser adapters are balanced.

Note: Samples loaded into the top and bottom rows of the TissueLyser adapter tend to be unequally homogenized.
Place the lid onto the TissueLyser adapters and load onto TissueLyser. Do not over-tighten the adapters in the TissueLyser.
Run the TissueLyser with a frequency of 25 Hz for 00:00:40 . Ensure tissue is dissociated and lysate is uniformly homogenous; if not, run the TissueLyser again with the same parameters.
Remove samples from the TissueLyser adapters. Let the homogenate rest for 00:03:00 at room temperature until the foam subsides.

In a fume hood, transfer the full volume of tissue homogenate and foam to a 2 mL Eppendorf Protein LoBind tube.
Let rest for 00:05:00 at room temperature.
Add 200 µL chloroform to each sample. Shake vigorously for 00:00:15 .
Incubate for 00:03:00 at room temperature.
Centrifuge at 12,000 x g for 00:15:00 at 4 °C .

Note: After centrifugation, heat the centrifuge to room temperature if the same centrifuge will be used in the later steps of this procedure.
Prepare 500 µL of 70% ethanol per sample.
Transfer the upper, aqueous phase to a new 1.5 mL DNA LoBind tube using a standard P200 pipette.

Note: After centrifugation, the sample separates into three phases: an upper, colorless, aqueous phase containing RNA; a white interphase; and a lower, red, organic phase. For tissues with an especially high fat content, an additional, clear phase may be visible below the red, organic phase. The volume of the aqueous phase should be approximately 500-600 µL.
Add 500 µL freshly prepared 70% ethanol to each sample. Mix thoroughly by vortexing for 30 seconds. Do not centrifuge.

Note: For samples with significantly more or less than 500 µL aqueous phase recovered in the previous step, add an equal volume of freshly prepared 70% ethanol.
Transfer up to 700 µL of each sample to an RNeasy Mini spin column placed in a 2 mL collection tube.

Note: The maximum volume capacity of an RNeasy Mini spin column is 700 µL. Do not overfill the spin column.
Centrifuge at 8,000 x g for 00:00:30 at room temperature.

Discard the flow-through from the collection tube into a designated hazardous waste container for QIAzol and chloroform waste. Place the spin column back into the 2 mL collection tube.

Note: After centrifugation, carefully remove the spin column from the collection tube so the spin column does not contact the flow-through. Be sure to empty the collection tube completely, and blot on a paper towel if necessary.
Transfer the remaining sample volume from step 33 to the spin column.
Centrifuge at 8,000 x g for 00:00:30 at room temperature.
Discard the flow-through from the collection tube into a designated hazardous waste container for QIAzol and chloroform waste. Place the spin column back into the 2 mL collection tube.

Note: After centrifugation, carefully remove the spin column from the collection tube so the spin column does not contact the flow-through. Be sure to empty the collection tube completely, and blot on a paper towel if necessary.

Note: After this step, the protocol can be performed at a lab bench cleaned with RNaseZap and 70% ethanol.
Add 700 µL Buffer RW1 to the spin column.

Centrifuge at 8,000 x g for 00:00:30 at room temperature.
Discard the flow-through from the collection tube into a designated hazardous waste container for QIAzol and chloroform waste. Place the spin column back into the 2 mL collection tube.

Note: After centrifugation, carefully remove the spin column from the collection tube so the spin column does not contact the flow-through. Be sure to empty the collection tube completely, and blot on a paper towel if necessary.
Add 500 µL Buffer RPE to the spin column.

Note: Buffer RPE is supplied as a concentrate. The kit uses RPE with a 80% final ethanol concentration. Before using, add the appropriate amount of ethanol (96-100%) to Buffer RPE as indicated on the bottle.
Centrifuge at 8,000 x g for 00:00:30 at room temperature.
Discard the flow-through from the collection tube into a designated hazardous waste container for QIAzol and chloroform waste. Place the spin column back into the 2 mL collection tube.

Note: After centrifugation, carefully remove the spin column from the collection tube so the spin column does not contact the flow-through. Be sure to empty the collection tube completely, and blot on a paper towel if necessary.
Add 500 µL Buffer RPE to the spin column.

Note: Buffer RPE is supplied as a concentrate. The kit uses RPE with a 80% final ethanol concentration. Before using, add the appropriate amount of ethanol (96-100%) to Buffer RPE as indicated on the bottle.
Centrifuge at 8,000 x g for 00:02:00 at room temperature.
Discard the flow-through from the collection tube into a designated hazardous waste container for QIAzol and chloroform waste.

Note: After centrifugation, carefully remove the spin column from the collection tube so the spin column does not contact the flow-through. Be sure to empty the collection tube completely, and blot on a paper towel if necessary.
Place the spin column into a new 2 mL collection tube.
Centrifuge at full speed (21,000 x g) for 00:01:00 at room temperature to dry the spin column.
Place the spin column into a new 1.5 mL DNA LoBind tube.
Add 30 µL RNase-free water to each sample.
Centrifuge at 8,000 x g for 00:01:00 at room temperature.
Add 30 µL RNase-free water to each sample.

Note: If high RNA concentration is required, use the eluate from the first elution instead of another volume of RNase-free water. If using the previous eluate, the RNA yield will be 15-30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.
Centrifuge at 8,000 x g for 00:01:00 at room temperature.
Discard spin column and keep RNA samples on ice.
Quantify by taking a single measurement on the Qubit Flex Fluorometer with the Qubit RNA BR Assay. Use 2 µL of RNA per measurement.
Quantify by taking a single measurement on the NanoDrop 8000 spectrophotometer. Use 2 µL of RNA per measurement.
Size on the Agilent 4200 TapeStation System with the RNA ScreenTape kit. Use 1 µL of RNA per measurement.
Figure 2. TapeStation RNA ScreenTape assay post-extraction

Store at -80 °C until ready for library preparation. At least 500 ng RNA is required for library preparation. If necessary, repeat extraction to achieve required mass for library preparation.
Part 1b: Extracting RNA from cultured cells using Qiagen RNeasy Plus Mini kit
Prepare an RNase-free workstation by wiping down surfaces, pipettes, tube racks, mini-centrifuges, and vortexes with 70% ethanol and RNaseZap. Use fresh tip boxes and tubes to minimize risk of contamination.
Prepare Buffer RLT Plus in a fume hood as follows:
  • Inspect Buffer RLT Plus to ensure it has not formed precipitates; if necessary, warm in 37°C water bath, then cool to room temperature before use
  • Aliquot the volume needed: 350 µL per sample for 5 million or fewer cells, or 600 µL per sample for 5-10 million cells
  • Supplement aliquot with 14.3 M 2-Mercaptoethanol (BME) by adding 10 µL BME for every 1 mL Buffer RLT Plus
  • Gently invert tube to mix
For 5 million or fewer cells, prepare 350 µL µL 70% ethanol per sample with nuclease-free water.
For 5-10 million cells, prepare 600 µL 70% ethanol per sample with nuclease-free water.

Note: To capture small RNAs, 100% ethanol will be used instead of 70% ethanol.
Obtain frozen cell pellet from -80 °C freezer. Thaw on ice for 00:05:00 .

Note: Cell input amount should not exceed 10 million cells so as not to exceed the spin column binding capacity. For new cell types and cells with high RNA content, start with fewer (e.g. 2-4 million) cells per reaction.

Note: This protocol purifies RNA molecules longer than 200 nucleotides. To extract total RNA including small RNAs, follow the modification at step 70. With the small RNA modification, do not exceed an input of 5 million cells.

Note: For fresh cell samples, harvest cells and centrifuge at 500 x g for 3-5 minutes at 4 °C to pellet cells. Remove the supernatant.
Flick mix 10x to loosen the cell pellet.
For 5 million or fewer cells, add 350 µL Buffer RLT Plus supplemented with BME to cell pellet.
For 5-10 million cells, add 600 µL Buffer RLT Plus supplemented with BME to cell pellet.
Vortex or pipette-mix thoroughly to resuspend.
Transfer sample to QIAshredder spin column in a 2 mL collection tube.
Centrifuge for 00:02:00 at full speed (21,000 x g).

Note: The flow-through contains the nucleic acid material - do not discard.
Transfer the flow-through to a gDNA Eliminator spin column in a 2 mL collection tube.
Centrifuge for 00:00:30 at 16,000 x g.

Note: If sample is left behind on the rim of the filter, repeat centrifugation.

Note: The flow through contains the nucleic acid material - do not discard.
Measure the flow-through volume and add 1 volume of 70% ethanol to the flow-through and pipette-mix 10x.
For 5 million or fewer cells, add 350 µL 70% ethanol to flow-through.
For 5-10 million cells, add 600 µL 70% ethanol to flow-through.

Note: To capture small RNAs, measure the flow-through volume and add 1.5 volumes of 100% ethanol to the flow-through and pipette-mix.
For 5 million or fewer cells, add 525 µL 100% ethanol to flow-through.
Transfer up to 700 µL of each sample to an RNeasy Mini spin column placed in a 2 mL collection tube.
Note: The maximum volume capacity of an RNeasy Mini spin column is 700 µL. Do not overfill the spin column.
Centrifuge at 16,000 x g for 00:00:15 at room temperature.
Discard the flow-through from the collection tube into a designated hazardous waste container for Buffer RLT Plus and BME waste. Place the spin column back into the 2 mL collection tube.

Note: After centrifugation, carefully remove the spin column from the collection tube so the spin column does not contact the flow-through. Be sure to empty the collection tube completely, and blot on a paper towel if necessary.
Transfer the remaining sample volume from step 71 to the spin column.
Centrifuge at 16,000 x g for 00:00:15 at room temperature.
Discard the flow-through from the collection tube into a designated hazardous waste container for Buffer RLT Plus and BME waste. Place the spin column back into the 2 mL collection tube.

Note: After centrifugation, carefully remove the spin column from the collection tube so the spin column does not contact the flow-through. Be sure to empty the collection tube completely, and blot on a paper towel if necessary.

Note: After this step, the protocol can be performed at a lab bench cleaned with RNaseZap and 70% ethanol.
Add 700 µL Buffer RW1 to the spin column.
Centrifuge at 16,000 x g for 00:00:15 at room temperature.
Discard the flow-through from the collection tube into a designated hazardous waste container for Buffer RLT Plus and BME waste. Place the spin column back into the 2 mL collection tube.

Note: After centrifugation, carefully remove the spin column from the collection tube so the spin column does not contact the flow-through. Be sure to empty the collection tube completely, and blot on a paper towel if necessary.
Add 500 µL Buffer RPE to the spin column.

Note: Buffer RPE is supplied as a concentrate. The kit uses RPE with a 80% final ethanol concentration. Before using, add the appropriate amount of ethanol (96-100%) to Buffer RPE as indicated on the bottle.
Centrifuge at 16,000 x g for 00:00:15 at room temperature.
Discard the flow-through from the collection tube into a designated hazardous waste container for Buffer RLT Plus and BME waste. Place the spin column back into the 2 mL collection tube.

Note: After centrifugation, carefully remove the spin column from the collection tube so the spin column does not contact the flow-through. Be sure to empty the collection tube completely, and blot on a paper towel if necessary.
Add 500 µL Buffer RPE to the spin column.

Note: Buffer RPE is supplied as a concentrate. The kit uses RPE with a 80% final ethanol concentration. Before using, add the appropriate amount of ethanol (96-100%) to Buffer RPE as indicated on the bottle.
Centrifuge at 16,000 x g for 00:02:00 at room temperature.
Discard the flow-through from the collection tube into a designated hazardous waste container for Buffer RLT Plus and BME waste. Place the spin column back into the 2 mL collection tube.

Note: After centrifugation, carefully remove the spin column from the collection tube so the spin column does not contact the flow-through. Be sure to empty the collection tube completely, and blot on a paper towel if necessary.
Place the spin column into a new 2 mL collection tube.
Centrifuge at full speed (21,000 x g) for 00:01:00 at room temperature to dry the spin column.
Place the spin column into a new 1.5 mL DNA LoBind tube.
Add 40 µL RNase-free water to each sample. Incubate for 30 seconds at room temperature.
Centrifuge at 16,000 x g for 00:01:00 at room temperature.
Add 40 µL RNase-free water to each sample.

Note: If high RNA concentration is required, use the eluate from the first elution instead of another volume of RNase-free water. If using the previous eluate, the RNA yield will be 15-30% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher.
Centrifuge at 16,000 x g for 00:01:00 at room temperature.
Discard spin column and keep RNA samples on ice.
Quantify by taking a single measurement on the Qubit Flex Fluorometer with the Qubit RNA BR Assay. Use 2 µL of RNA per measurement.
Quantify by taking a single measurement on the NanoDrop 8000 spectrophotometer. Use 2 µL of RNA per measurement.
Size on the Agilent 4200 TapeStation System with the RNA ScreenTape kit. Use 1 µL of RNA per measurement.
Store at -80 °C until ready for library preparation. At least 500 ng RNA is required for library preparation. If necessary, repeat extraction to achieve required mass for library preparation.

Part 2a: cDNA-PCR sequencing V14 (SQK-PCS114) library preparation
1h 5m
Note: For automated library preparation, see Part 2b: cDNA-PCR sequencing V14 (SQK-PCS114) library preparation on Hamilton NGS STAR.

Note: For a lower RNA input protocol, see Part 2c: Modified cDNA-PCR sequencing V14 (SQK-PCS114) library preparation for 100 ng total RNA input

Thaw the following reagents at room temperature, then pipette-mix, spin down, and place on ice:
  • cDNA RT Adapter (CRTA)
  • Annealing Buffer (AB)
  • RT Primer (RTP)
  • Strand Switching Primer II (SSPII)
  • 10 mM dNTP solution
  • cDNA Primer (cPRM)
  • Elution Buffer (EB)
Thaw the following reagents at room temperature, then mix by vortexing, spin down, and place on ice:
  • StickTogether DNA Ligase Buffer
  • Maxima H Minus 5x RT Buffer
  • Short Fragment Buffer (SFB)
Place the following reagents at room temperature:
  • RNAClean XP beads
  • AMPure XP beads
Prepare an RNase-free workstation by wiping down surfaces, pipettes, tube racks, minicentrifuges, and vortexes with 70% ethanol and RNaseZap. Use fresh tip boxes and tubes to minimize risk of contamination.
Prepare 1:100 SIRV-Set 4 dilution as follows:
  • Upon receipt of SIRV-Set 4, thaw on ice, aliquot 18 x 0.5 µL aliquots into DNA LoBind tubes, and freeze at -20 °C
  • Immediately prior to spike-in to RNA samples, add 49.5 µL of nuclease-free water to a 0.5 µL aliquot of SIRV-Set 4, gently pipette-mix 10x, and spin down
  • Discard any remaining diluted SIRV-Set 4; freeze-thaw of diluted SIRVs may have an impact on RNA integrity and concentration
Prepare 500 ng total RNA in 12.2 µL nuclease-free water in a 0.2 mL thin-walled PCR tube or tube strip.
Add 2.8 µL diluted SIRV-Set 4 to each sample.

Note: SIRV-Set 4 volume is calculated based on total RNA input, estimated fraction of mRNA, and dilution factor. See Lexogen for more details.
Prepare the following annealing master mix, adjusting the reagent volumes for the number of samples being processed:
  • 1 µL cDNA RT Adapter
  • 1 µL Annealing Buffer

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Prepare the annealing master mix with a 10% overage.
Add 2 µL annealing master mix to each sample. Pipette-mix 10x and spin down.
Incubate samples at 60 °C for 00:05:00 in a thermocycler with the lid temperature set to 85 °C .
Incubate samples at room temperature for 00:05:00 .
Prepare the following ligation master mix, adjusting the reagent volumes for the number of samples being processed:
  • 19 µL StickTogether DNA Ligase Buffer
  • 1 µL T3 DNA Ligase
  • 1 µL RNaseOUT

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Keep T3 DNA Ligase in the freezer until use and return promptly.

Note: Prepare the ligation master mix with a 10% overage.
Add 21 µL ligation master mix to each sample. Pipette-mix 10x and spin down.
Incubate samples at room temperature for 00:10:00 .
Prepare the following digestion master mix, adjusting the reagent volumes for the number of samples being processed:
  • 1 µL Lambda Exonuclease
  • 1 µL USER (Uracil-Specific Excision Reagent)

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Keep enzyme mixes in the freezer until use and return promptly.

Note: Prepare the digestion master mix with a 10% overage.
Add 2 µL digestion master mix to each sample. Pipette-mix 10x and spin down.
Incubate samples at 37 °C for 00:05:00 in a thermocycler with the lid temperature set to 85 °C .
Transfer samples to new 1.5 mL DNA LoBind tubes.
Resuspend RNAClean XP beads by vortexing.
Add 72 µL RNAClean XP beads. Flick mix 10x and spin down.
Incubate at room temperature for 00:05:00 .
Spin down samples and place on the magnetic rack. Wait until supernatant is clear and colorless, about 00:02:00 .
Keep the tube on the magnetic rack and pipette off the supernatant.
With the samples remaining on the magnetic rack, wash the samples with Short Fragment Buffer (SFB):
  • Add 100 µL SFB
  • Rotate the tube 180° and wait until the beads migrate back to the magnet
  • Rotate the tube 180° (back to the starting position) and wait until the beads migrate back to the magnet
  • Remove the supernatant, pipetting on the opposite wall to avoid disturbing the pellet
Repeat the previous step.
Spin down samples and place on the magnetic rack. Pipette off any residual SFB.
Allow to dry for ~00:00:30 , but do not overdry to the point of cracking.
Remove tubes from the magnetic rack. Add 12 µL nuclease-free water. Flick mix 10x and spin down.
Incubate at room temperature for 00:10:00 .
Spin down samples and place on the magnetic rack. Wait until supernatant is clear and colorless, about 00:02:00 .
Transfer 12 µL of eluate into a new 0.2 mL thin-walled PCR tube or tube strip.
Prepare the following priming master mix, adjusting the reagent volumes for the number of samples being processed:
  • 1 µL RT Primer
  • 1 µL dNTPs

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Prepare the priming master mix with a 10% overage.
Add 2 µL priming master mix to each sample. Pipette-mix 10x and spin down.
Incubate at room temperature for 00:05:00 .
Prepare the following strand-switching master mix, adjusting the reagent volumes for the number of samples being processed:
  • 4.5 µL Maxima H Minus 5x RT Buffer
  • 1 µL RNaseOUT
  • 2 µL Strand Switching Primer II

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Keep enzyme mixes in the freezer until use and return promptly.

Note: Prepare the strand-switching master mix with a 10% overage.
Add 7.5 µL strand-switching master mix to each sample. Pipette-mix 10x and spin down.
Incubate at 42 °C for 00:02:00 in a thermocycler with the lid temperature set to 105 °C .
Add 1 µL Maxima H Minus Reverse Transcriptase to each sample. Pipette-mix 10x and spin down.

Note: Keep Maxima H Minus Reverse Transcriptase in the freezer until use and return promptly.
Incubate in a thermocycler using the following protocol:
  • 42 °C for 01:00:00 (1x) - Reverse transcription and strand switching
  • 85 °C for 00:05:00 (1x) - Heat inactivation
  • Hold at 4 °C
  • Lid temperature set to 105 °C

Note: It is possible to store samples at -20 °C overnight at this step.
1h 5m
Spin down samples in minicentrifuge.
Prepare the following PCR master mix for 4x PCR reactions per sample, adjusting the reagent volumes for the number of samples being processed:
  • 25 µL Ex Premier DNA Polymerase
  • 0.5 µL cDNA Primer (cPRM)
  • 19.5 µL nuclease-free water

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Keep Ex Premier DNA Polymerase in the freezer until use and return promptly.

Note: Prepare the PCR master mix with a 10% overage.
Aliquot 45 µL of the PCR master mix into new 0.2 mL thin-walled PCR tubes or tube strips to prepare 4x PCR reactions per sample.
Add 5 µL cDNA sample to each PCR reaction, splitting the 22.5 µL reverse transcription reaction across 4x PCR reactions. Pipette-mix 10x and spin down.
Incubate in a thermoycler using the following protocol:
  • 98 °C for 00:00:10 (22x) - Initial denaturation
  • 68 °C for 00:03:00 (22x) - Final extension
  • Hold at 4 °C
  • Lid temperature set to 105 °C

Note: When using the Bio-Rad thermocycler, the “number of repeats” parameter does not include the first cycle. As such, for 22 cycles, the protocol should be programmed to repeat 21 times.
Add 1 µL Thermolabile Exonuclease I to each PCR reaction. Pipette-mix 10x and spin down.

Note: Keep Thermolabile Exonuclease I in the freezer until use and return promptly.
Incubate in a thermocycler using the following protocol:
  • 37 °C for 00:05:00 (1x)
  • 80 °C for 00:02:00 (1x)
  • Lid temperature set to 105 °C
Pool the 4x PCR reactions into a new 1.5 mL DNA LoBind tube.
Resuspend AMPure XP beads by vortexing.
Add 140 µL AMPure XP beads. Flick mix 10x and spin down.
Incubate at room temperature for 00:05:00 .
Prepare 1 mL of fresh 80% ethanol per sample in nuclease-free water.
Spin down samples and place on the magnetic rack. Wait until supernatant is clear and colorless, about 00:02:00 .
Keep the tube on the magnetic rack and pipette off the supernatant.
With the samples remaining on the magnetic rack, add 500 µL of 80% ethanol. Pipette on the opposite wall to avoid disturbing the pellet. After 00:00:05 , remove the ethanol. Do not resuspend the beads in ethanol.
Repeat the previous step.
Spin down samples and place on the magnetic rack. Pipette off any residual ethanol.
Allow to dry for ~00:00:30 , but do not overdry to the point of cracking.
Remove tubes from the magnetic rack. Add 25 µL Elution Buffer (EB). Flick mix 10x and spin down.
Incubate at room temperature for 00:10:00 .
Spin down samples and place on the magnetic rack. Wait until supernatant is clear and colorless, about 00:02:00 .
Transfer 25 µL of eluate into a new 1.5 mL DNA LoBind tube.
Quantify by taking a single measurement on the Qubit Flex Fluorometer with the Qubit 1x dsDNA HS Assay. Use 1 µL of cDNA per measurement.
Size on the Agilent 4200 TapeStation System with the D5000 DNA ScreenTape assay. Use 1 µL of cDNA per measurement. The expected average size for samples post-PCR is ~3kb.
Figure 3. TapeStation D5000 ScreenTape assay post-library preparation

Store at 4 °C until ready for adapter addition and flow cell loading. At least 50 fmol cDNA is required for adapter addition and flow cell loading. If necessary, repeat library preparation to achieve required loading femtomoles.
Part 2b: cDNA-PCR sequencing V14 (SQK-PCS114) library preparation on Hamilton NGS STAR
Note: For manual library preparation, see Part 2a: cDNA-PCR sequencing V14 (SQK-PCS114) library preparation.

Thaw the following reagents at room temperature, then pipette-mix, spin down, and place on ice:
  • cDNA RT Adapter (CRTA)
  • Annealing Buffer (AB)
  • RT Primer (RTP)
  • Strand Switching Primer II (SSPII)
  • 10 mM dNTP solution
  • cDNA Primer (cPRM)
  • Elution Buffer (EB)
Thaw the following reagents at room temperature, then mix by vortexing, spin down, and place on ice:
  • StickTogether DNA Ligase Buffer
  • Maxima H Minus 5x RT Buffer
  • Short Fragment Buffer (SFB)
Place the following reagents at room temperature:
  • RNAClean XP beads
  • AMPure XP beads
Prepare an RNase-free workstation by wiping down surfaces, pipettes, tube racks, minicentrifuges, and vortexes with 70% ethanol and RNaseZap. Use fresh tip boxes and tubes to minimize risk of contamination.
Prepare 1:100 SIRV-Set 4 dilution as follows:
  • Upon receipt of SIRV-Set 4, thaw on ice, aliquot 18 x 0.5 µL aliquots into DNA LoBind tubes, and freeze at -20 °C
  • Immediately prior to spike-in to RNA samples, add 49.5 µL of nuclease-free water to a 0.5 µL aliquot of SIRV-Set 4, gently pipette-mix 10x, and spin down
  • Discard any remaining diluted SIRV-Set 4; freeze-thaw of diluted SIRVs may have an impact on RNA integrity and concentration
Prepare 500 ng total RNA in 17.2 µL nuclease-free water in a Bio-Rad Hard-Shell PCR Plate.

Note: Samples must be processed in multiples of 8. If preparing less samples than a multiple of 8, fill remaining wells with 20 µL nuclease-free water.
Add 2.8 µL diluted SIRV-Set 4 to each sample.

Note: SIRV-Set 4 volume is calculated based on total RNA input, estimated fraction of mRNA, and dilution factor. See Lexogen for more details.
Power on the NGS STAR system:
  • Power on the NGS STAR instrument with the power switch on the left side of the instrument.
  • Power on the CPAC cooler underneath the instrument on the right side, on top of the computer, with the power switch on the back.
  • Power on the HHS controller underneath the instrument on the left side, with the power switch on the front.
  • Power on the ODTC controller underneath the instrument on the left side, with the power switch on the front.
  • Power on the filter and fan on top of the instrument by pressing the fan button on the left side. Once initialized, use the stylus to set the fan power to 50%, with the digital knob pointing up.
Empty Hamilton tip waste containers.
Launch the PCS114 protocol in the Hamilton Method Manager.

Note: Ensure the deck layout of the NGS STAR matches the protocol being run.

Note: Daily and weekly maintenance must be performed prior to beginning the protocol. Follow manufacturer guidelines for preventive maintenance and cleaning procedures.
Select “Process 1: Reverse Transcription and Strand Switching” as the start process.
Figure 4. Select start process

Select “Process 2: Amplification” as the stop process.
Figure 5. Select stop process

Select the On-deck Thermal Cycler (ODTC) status.
Figure 6. Select ODTC

Select the number of samples being processed.
Figure 7. Select number of samples

Prepare the reagent master mixes in Sarstedt tubes according to the instructions in the method. Gently pipette-mix 10x and spin down. Avoid pipetting bubbles.
Figure 8. Prepare reagent master mixes

Note: To ensure optimal reagent stability and limit the time reagents are on the NGS STAR deck, prepare the reagent master mixes immediately prior to use, and load them as prompted during the method.
Load PCR ComfortLids onto the NGS STAR deck according to the instructions in the method.
Figure 9. Load PCR ComfortLids

Load plates onto the NGS STAR deck according to the instructions in the method.

Figure 10. Load plates

Load 50 µL and 1000 µL tips onto the NGS STAR tip carriers according to the instructions in the method. Use the tip count screen to input loaded 50 µL and 1000 µL tips.
Figure 11. Load 50 µL and 1000 µL tips

Figure 12. Select loaded 50 µL tips

Figure 13. Select loaded 1000 µL tips

Note: Method should be started with full tip racks to avoid unnecessary errors.
Load 300 µL tips onto the NGS STAR tip carriers according to the instructions in the method. Use the tip count screen to input loaded 300 µL tips.

Figure 14. Load 300 µL tips

Figure 15. Select loaded 300 µL tips

Note: Method should be started with full tip racks to avoid unnecessary errors.
Load reagents in 20 mL reagent troughs in the first reagent trough carrier according to the instructions in the method.

Figure 16. Load first reagent trough carrier

Load reagents in 20 mL reagent troughs in the second reagent trough carrier according to the instructions in the method.
Figure 17. Load second reagent trough carrier

Load prepared sample plate with 20 µL total RNA per well and 80% ethanol in a 300 mL reservoir onto the NGS STAR according to the instructions in the method.
Figure 18. Load sample plate carrier

Note: Ensure the magnetic stand is placed on the carrier in the 5th position.
Load the reagent master mix tubes onto the CPAC cooler according to the instructions in the method. To start the method, load the Annealing Mix, Ligation Mix, and Digestion Mix.

Figure 19. Load reagent master mix tubes

Note: Enzyme mixes should be spun down and placed on the NGS STAR deck with the caps removed. Take care to avoid pipetting bubbles in the tubes to avoid inaccurate dispensing.

Note: To ensure optimal reagent stability and limit the time reagents are on the NGS STAR deck, prepare the reagent master mixes immediately prior to use, and load them as prompted during the method.
Close the NGS STAR door and press 'Ok.' The protocol will now begin.
Figure 20. Loading complete dialog

When prompted during the method (after the first bead cleanup), open the NGS STAR door and load the RT Primer Mix, SSPII Primer Mix, and Maxima H Minus RT onto the CPAC cooler. Close the NGS STAR door and press 'Ok.'
Figure 21. Reverse transcription reagent loading dialog

When prompted during the method (after reverse transcription incubation), open the NGS STAR door and load the PCR mix onto the CPAC cooler. Close the NGS STAR door and press ‘Ok.’
Figure 22. PCR mix loading dialog

When prompted during the method (after PCR incubation), open the NGS STAR door and load the Thermolabile Exonuclease onto the CPAC cooler. Close the NGS STAR door and press ‘Ok.’
Figure 23. Thermolabile Exonuclease loading dialog

When prompted at the end of the run, remove the sample plate with cDNA library from the NGS STAR. Discard the reagent troughs and enzyme mix tubes.
Figure 24. Run completion dialog

Quantify by taking a single measurement on the Qubit Flex Fluorometer with the Qubit 1x dsDNA HS Assay. Use 1 µL of cDNA per measurement.
Size on the Agilent 4200 TapeStation System with the D5000 DNA ScreenTape assay. Use 1 µL of cDNA per measurement. The expected average size for samples post-PCR is ~3kb.
Figure 25. TapeStation D5000 ScreenTape assay post-library preparation for human brain sampleL

Seal the plate containing cDNA libraries with a foil seal using a seal roller, ensuring all wells are completely sealed. Spin down if necessary. If desired, transfer cDNA libraries into new 1.5 mL DNA LoBind tubes.
Store at 4 °C until ready for adapter addition and flow cell loading. At least 50 fmol cDNA is required for adapter addition and flow cell loading. If necessary, repeat library preparation to achieve required loading moles.
Part 3a: Adapter addition
Note: For automated adapter addition, see Part 3b: Adapter addition on Hamilton NGS STAR.

Thaw the following reagents at room temperature, then mix by vortexing, spin down, and place on ice:
  • Sequencing Buffer (SB)
  • Library Beads (LIB)
  • Elution Buffer (EB)
  • Flow Cell Flush (FCF)
  • Flow Cell Tether (FCT)
Gently mix the following reagents by pipetting, spin down, and place on ice:
  • Rapid Adapter (RA)
  • Adapter Buffer (ADB)
Take flow cells out of the fridge. Allow to sit at room temperature for 00:20:00 .

Note: Condensation can form on the flow cell in humid environments. Inspect the gold connector pins on the top and underside of the flow cell for condensation and wipe off with a lint-free wipe if any is observed. Ensure the heat pad (black pad) is present on the underside of the flow cell.
Prepare 50 fmol cDNA library in 31 µL Elution Buffer in a new 1.5 mL DNA LoBind tube. Flick mix 10x and spin down.
Prepare the following rapid adapter master mix, adjusting the reagent volumes for the number of samples being processed. Pipette gently 10x to mix. Avoid pipetting bubbles.
  • 0.3 µL Rapid Adapter
  • 0.7 µL Adapter Buffer

Note: Prepare the rapid adapter master mix with a 10% overage.
Add 1 µL rapid adapter master mix to each sample. Flick mix 10x and spin down.
Incubate at room temperature for 00:05:00
Add 100 µL  Sequencing Buffer to each sample.
Add 68 µL  Library Beads to each sample.

Note: Vortex Library Beads immediately prior to pipetting to ensure complete resuspension.
Proceed immediately to flow cell loading.
Part 3b: Adapter addition on Hamilton NGS STAR
Note: For manual adapter addition, see Part 3a: Adapter addition.

Thaw the following reagents at room temperature, then mix by vortexing, spin down, and place on ice:
  • Sequencing Buffer (SB)
  • Library Beads (LIB)
  • Elution Buffer (EB)
  • Flow Cell Flush (FCF)
  • Flow Cell Tether (FCT)
Gently mix the following reagents by pipetting, spin down, then place on ice:
  • Rapid Adapter (RA)
  • Adapter Buffer (ADB)
If automated normalization is enabled, obtain Bio-Rad Hard-Shell PCR Plate with at least 50 fmol per. Prepare input file with sample volumes and concentrations. Spin down plate for XX seconds.

If automated normalization is not enabled, prepare 50 fmol cDNA library in 31 µL Elution Buffer in a Bio-Rad Hard-Shell PCR Plate.
Take flow cells out of the fridge. Allow to sit at room temperature for 00:20:00 .

Note: Condensation can form on the flow cell in humid environments. Inspect the gold connector pins on the top and underside of the flow cell for condensation and wipe off with a lint-free wipe if any is observed. Ensure the heat pad (black pad) is present on the underside of the flow cell.
Launch the PCS114 protocol in the Hamilton Method Manager.

Note: Ensure the deck layout of the NGS STAR matches the protocol being run.

Note: Daily and weekly maintenance must be performed prior to beginning the protocol. Follow manufacturer guidelines for preventive maintenance and cleaning procedures.
Select “Process 3: Adapter Addition” as the start process.
Figure 26. Select start process

Select “Process 3: Adapter Addition” as the stop process.
Figure 27. Select stop process

Select the number of samples being processed.
Figure 28. Select number of samples

Select if automated sample normalization should be performed.
Figure 29. Automated sample normalization dialog

Select the input normalization file worklist.
Figure 30. Input normalization file dialog

Prepare the rapid adapter master mix according to the instructions in the method. Gently pipette-mix 10x and spin down. Avoid pipetting bubbles.
Figure 31. Prepare rapid adapter master mix

Load 50 µL tips onto the NGS STAR tip carriers according to the instructions in the method. Use the tip count screen to input loaded 50 µL tips.
Figure 32. Load 50 µL tips

Figure 33. Select loaded 50 µL tips

Load 300 µL tips onto the NGS STAR tip carriers according to the instructions in the method. Use the tip count screen to input loaded 300 µL tips.
Figure 34. Load 300 µL tips

Figure 35. Select loaded 300 µL tips

Load reagents in 20 mL reagent troughs in the second reagent trough carrier according to the instructions in the method.
Figure 36. Load second reagent trough carrier

Note: Vortex Library Beads immediately prior to pipetting to ensure complete resuspension.
Load empty 1.5 mL DNA LoBind tubes onto the first 32-tube carrier according to the instructions in the method.
Figure 37. Load sample tube carrier

Note: Ensure tubes are open with the lids at a 90 degree angle, facing up, and not blocking the pipetting channels from entering the tube. Failure to open the tubes correctly could cause the pipetting channels to crash into the tubes and abort the method.
If automated sample normalization is selected, load sample plate with cDNA libraries onto the NGS STAR according to the instructions in the method. Ensure more than 50 fmol is available in each well for processing.

If automated sample normalization is not selected, load prepared sample plate with 50 fmol cDNA library in 31 µL Elution Buffer per well onto the NGS STAR according to the instructions in the method.
Figure 38. Load sample plate carrier

Load reagent tubes onto the CPAC cooler according to the instructions in the method.
Figure 39. Load reagents onto CPAC

Note: Reagents should be spun down and placed on the NGS STAR deck with the caps removed. Take care to avoid pipetting bubbles in the tubes to avoid inaccurate dispensing.
Close the NGS STAR door and press 'Ok.' The protocol will now begin.
Figure 40. Loading complete dialog

When prompted at the end of the run, remove the sample tubes with ready-to-load cDNA libraries from the NGS STAR. Discard the reagent troughs and enzyme mix tubes. If automated sample normalization is selected, re-seal the plate with remaining cDNA libraries and store at 4 °C .
Figure 41. Run completion dialog

Proceed immediately to flow cell loading.
Part 4: Loading the PromethION Flow Cell
Load flow cells into the PromethION docking ports. Perform flow cell check prior to flow cell priming.

Note: Only flow cells with a starting pore count greater than 6500 pores should be used for sequencing runs. Flow cells with starting pore counts less than 6500 pores can be used to generate additional sequencing data after an initial run.
Prepare the flow cell priming mix by combining 1170 µL Flow Cell Flush and30 µL  Flow Cell Tether per sample. Mix by vortexing.
Slide the inlet port cover clockwise to open. Draw back a small volume to remove any air bubbles:
  • Set a P1000 pipette tip to 200 µL
  • Insert the tip into the inlet port
  • Turn the wheel until dial shows 220-230 µL, until a small volume of buffer enters the pipette tip
Note: Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µL, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
Load 500 µL  of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles.
Wait 00:05:00 .
Load 500 µL  of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles.
Mix the prepared library gently by pipetting up and down 5x just prior to loading.
Load 200 µL  of library into the inlet port using a P1000 pipette.
Close the valve to seal the inlet port. Install the light shield. Close the PromethION door.
Wait 00:10:00  before initiating sequencing run in MinKNOW:
  • Navigate to the start page and click "Start sequencing"
  • Fill in the experiment name and sample ID, select the flow cell position, and load run configuration preset (if applicable)
  • Select the sequencing kit used in the library preparation (SQK-PCS114) on the Kit page
  • Configure sequencing and output parameters - Fast basecalling model, 72 hour run time, basecalled output off, raw reads output .POD5, and minimum Q score of 8
  • Ensure there is enough storage space on the computer with at least 2 Tb of available space per cDNA sequencing run
  • Click "Start" to initiate the sequencing run
Within the first hour of sequencing, pay attention to pore occupancy and pore scan results. If necessary to resolve sequencing issues related to sample or flow cell quality:
  1. Perform pore scan on flow cell and note if it resolves pore count issues.
  2. Stop the sequencing run, remove the flow cell from the PromethION, and insert it again into a different position. Restart the run and note if it resolves pore count issues.
  3. Stop the sequencing run. Recover cDNA library and load onto a new flow cell, following protocol from step 227.
Following 72 hours of sequencing, the sample should yield a data output ~70 million reads and ~150 Gb with an N50 ~2.5-3 kb.

Figure 42. Read length distribution for brain tissue sample with a 72 hour sequencing run

Figure 43. PromethION pore scan results for brain tissue sample with a 72 hour sequencing run

Part 2c: Modified cDNA-PCR sequencing V14 (SQK-PCS114) library preparation for 100 ng total RNA input
Note: For the standard RNA input protocol, see Part 2a: cDNA-PCR sequencing V14 (SQK-PCS114) library preparation

Thaw the following reagents at room temperature, then pipette-mix, spin down, and place on ice:
  • cDNA RT Adapter (CRTA)
  • Annealing Buffer (AB)
  • RT Primer (RTP)
  • Strand Switching Primer II (SSPII)
  • 10 mM dNTP solution
  • cDNA Primer (cPRM)
  • Elution Buffer (EB)
Thaw the following reagents at room temperature, then mix by vortexing, spin down, and place on ice:
  • StickTogether DNA Ligase Buffer
  • Maxima H Minus 5x RT Buffer
  • Short Fragment Buffer (SFB)
Place the following reagents at room temperature:
  • RNAClean XP beads
  • AMPure XP beads
Prepare an RNase-free workstation by wiping down surfaces, pipettes, tube racks, minicentrifuges, and vortexes with 70% ethanol and RNaseZap. Use fresh tip boxes and tubes to minimize risk of contamination.
Prepare 1:100 SIRV-Set 4 dilution as follows:
  • Upon receipt of SIRV-Set 4, thaw on ice, aliquot 18 x 0.5 µL aliquots into DNA LoBind tubes, and freeze at -20 °C
  • Immediately prior to spike-in to RNA samples, add 49.5 µL of nuclease-free water to a 0.5 µL aliquot of SIRV-Set 4, gently pipette-mix 10x, and spin down
  • Discard any remaining diluted SIRV-Set 4; freeze-thaw of diluted SIRVs may have an impact on RNA integrity and concentration
Prepare 100 ng total RNA in 19.44 µL nuclease-free water in a 0.2 mL thin-walled PCR tube or tube strip.
Add 0.56 µL diluted SIRV-Set 4 to each sample.

Note: SIRV-Set 4 volume is calculated based on total RNA input, estimated fraction of mRNA, and dilution factor. See Lexogen for more details.
Prepare the following annealing master mix, adjusting the reagent volumes for the number of samples being processed:
  • 1 µL cDNA RT Adapter
  • 1 µL Annealing Buffer

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Prepare the annealing master mix with a 10% overage.
Add 2 µL annealing master mix to each sample. Pipette-mix 10x and spin down.
Incubate samples at 60 °C for 00:05:00 in a thermocycler with the lid temperature set to 85 °C .
Incubate samples at room temperature for 00:05:00 .
Prepare the following ligation master mix, adjusting the reagent volumes for the number of samples being processed:
  • 24 µL StickTogether DNA Ligase Buffer
  • 1 µL T3 DNA Ligase
  • 1 µL RNaseOUT

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Keep T3 DNA Ligase in the freezer until use and return promptly.

Note: Prepare the ligation master mix with a 10% overage.
Add 26 µL ligation master mix to each sample. Pipette-mix 10x and spin down.
Incubate samples at room temperature for 00:10:00 .
Prepare the following digestion master mix, adjusting the reagent volumes for the number of samples being processed:
  • 1 µL Lambda Exonuclease
  • 1 µL USER (Uracil-Specific Excision Reagent)

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Keep enzyme mixes in the freezer until use and return promptly.

Note: Prepare the digestion master mix with a 10% overage.
Add 2 µL digestion master mix to each sample. Pipette-mix 10x and spin down.
Incubate samples at 37 °C for 00:05:00 in a thermocycler with the lid temperature set to 85 °C .
Transfer samples to new 1.5 mL DNA LoBind tubes.
Resuspend RNAClean XP beads by vortexing.
Add 90 µL RNAClean XP beads. Flick mix 10x and spin down.
Incubate at room temperature for 00:05:00 .
Spin down samples and place on the magnetic rack. Wait until supernatant is clear and colorless, about 00:02:00 .
Keep the tube on the magnetic rack and pipette off the supernatant.
With the samples remaining on the magnetic rack, wash the samples with Short Fragment Buffer (SFB):
  • Add 150 µL SFB
  • Rotate the tube 180° and wait until the beads migrate back to the magnet
  • Rotate the tube 180° (back to the starting position) and wait until the beads migrate back to the magnet
  • Remove the supernatant, pipetting on the opposite wall to avoid disturbing the pellet
Repeat the previous step.
Spin down samples and place on the magnetic rack. Pipette off any residual SFB.
Allow to dry for ~00:00:30 , but do not overdry to the point of cracking.
Remove tubes from the magnetic rack. Add 12 µL nuclease-free water. Flick mix 10x and spin down.
Incubate at room temperature for 00:10:00 .
Spin down samples and place on the magnetic rack. Wait until supernatant is clear and colorless, about 00:02:00 .
Transfer 12 µL of eluate into a new 0.2 mL thin-walled PCR tube or tube strip.
Prepare the following priming master mix, adjusting the reagent volumes for the number of samples being processed:
  • 1 µL RT Primer
  • 1 µL dNTPs

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Prepare the priming master mix with a 10% overage.
Add 2 µL priming master mix to each sample. Pipette-mix 10x and spin down.
Incubate at room temperature for 00:05:00 .
Prepare the following strand-switching master mix, adjusting the reagent volumes for the number of samples being processed:
  • 4.5 µL Maxima H Minus 5x RT Buffer
  • 1 µL RNaseOUT
  • 2 µL Strand Switching Primer II

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Keep enzyme mixes in the freezer until use and return promptly.

Note: Prepare the strand-switching master mix with a 10% overage.
Add 7.5 µL strand-switching master mix to each sample. Pipette-mix 10x and spin down.
Incubate at 42 °C for 00:02:00 in a thermocycler with the lid temperature set to 105 °C .
Add 1 µL Maxima H Minus Reverse Transcriptase to each sample. Pipette-mix 10x and spin down.

Note: Keep Maxima H Minus Reverse Transcriptase in the freezer until use and return promptly.
Incubate in a thermocycler using the following protocol:
  • 42 °C for 01:00:00 (1x) - Reverse transcription and strand switching
  • 85 °C for 00:05:00 (1x) - Heat inactivation
  • Hold at 4 °C
  • Lid temperature set to 105 °C

Note: It is possible to store samples at -20 °C overnight at this step.
Spin down samples in minicentrifuge.
Prepare the following PCR master mix for 4x PCR reactions per sample, adjusting the reagent volumes for the number of samples being processed:
  • 25 µL Ex Premier DNA Polymerase
  • 0.5 µL cDNA Primer (cPRM)
  • 19.5 µL nuclease-free water

Pipette gently 10x to mix. Avoid pipetting bubbles.

Note: Keep Ex Premier DNA Polymerase in the freezer until use and return promptly.

Note: Prepare the PCR master mix with a 10% overage.
Aliquot 45 µL of the PCR master mix into new 0.2 mL thin-walled PCR tubes or tube strips to prepare 4x PCR reactions per sample.
Add 5 µL cDNA sample to each PCR reaction, splitting the 22.5 µL reverse transcription reaction across 4x PCR reactions. Pipette-mix 10x and spin down.
Incubate in a thermoycler using the following protocol:
  • 98 °C for 00:00:10 (5x) - Initial denaturation
  • 50 °C for 00:00:30 (5x) - Initial annealing
  • 68 °C for 00:03:00 (5x) - Initial extension
  • 98 °C for 00:00:10 (18x) - Denaturation
  • 68 °C for 00:03:00 (18x) - Annealing and extension
  • Hold at 4 °C
  • Lid temperature set to 105 °C

Note: When using the Bio-Rad thermocycler, the “number of repeats” parameter does not include the first cycle. As such, for 5 cycles and 18 cycles, the protocol should be programmed to repeat 4 times and 17 times, respectively.
Add 1 µL Thermolabile Exonuclease I to each PCR reaction. Pipette-mix 10x and spin down.

Note: Keep Thermolabile Exonuclease I in the freezer until use and return promptly.
Incubate in a thermocycler using the following protocol:
  • 37 °C for 00:05:00 (1x)
  • 80 °C for 00:02:00 (1x)
  • Lid temperature set to 105 °C
Pool the 4x PCR reactions into a new 1.5 mL DNA LoBind tube.
Resuspend AMPure XP beads by vortexing.
Add 245 µL AMPure XP beads. Flick mix 10x and spin down.
Incubate at room temperature for 00:05:00 .
Prepare 1 mL of fresh 80% ethanol per sample in nuclease-free water.
Spin down samples and place on the magnetic rack. Wait until supernatant is clear and colorless, about 00:02:00 .
Keep the tube on the magnetic rack and pipette off the supernatant.
With the samples remaining on the magnetic rack, add 500 µL of 80% ethanol. Pipette on the opposite wall to avoid disturbing the pellet. After 00:00:05 , remove the ethanol. Do not resuspend the beads in ethanol.
Repeat the previous step.
Spin down samples and place on the magnetic rack. Pipette off any residual ethanol.
Allow to dry for ~00:00:30 , but do not overdry to the point of cracking.
Remove tubes from the magnetic rack. Add 30 µL Elution Buffer (EB). Flick mix 10x and spin down.

Note: If a higher concentration is necessary, add 20 µL Elution Buffer (EB).
Incubate at room temperature for 00:10:00 .
Spin down samples and place on the magnetic rack. Wait until supernatant is clear and colorless, about 00:02:00 .
Transfer 30 µL of eluate into a new 1.5 mL DNA LoBind tube.
Quantify by taking a single measurement on the Qubit Flex Fluorometer with the Qubit 1x dsDNA HS Assay. Use 1 µL of cDNA per measurement.
Size on the Agilent 4200 TapeStation System with the D5000 DNA ScreenTape assay. Use 1 µL of cDNA per measurement. The expected average size for samples post-PCR is ~3kb.
Figure 44. TapeStation D5000 ScreenTape assay post-library preparation

Store at 4 °C until ready for adapter addition and flow cell loading. At least 50 fmol cDNA is required for adapter addition and flow cell loading. If necessary, repeat library preparation to achieve required loading femtomoles.
Protocol references
  1. TissueLyser III User Manual. QIAGEN, 2023, https://www.qiagen.com/us/resources/resourcedetail?id=c41836ee-0782-486c-9f1e-61f21f955588&lang=en.
  2. RNeasy Lipid Tissue Mini Handbook. QIAGEN, 2018. https://www.qiagen.com/us/resources/resourcedetail?id=7f13ac1a-841d-4e9b-b39d-42fe71b3d585&lang=en.
  3. RNeasy Plus Mini Handbook. QIAGEN, 2020, https://www.qiagen.com/us/resources/resourcedetail?id=16b8f578-d192-4613-ae32-8e02e0b0fa77&lang=en.
  4. cDNA-PCR Sequencing V14 (SQK-PCS114). Oxford Nanopore Technologies, 2025, https://nanoporetech.com/document/pcr-cdna-sequencing-v14-sqk-pcs114.