Aug 20, 2025
prime-seq 2
- Felix Pförtner1,
- Eva Briem1,
- Wolfgang Enard1,
- Daniel Richter1
- 1Ludwig-Maximilians-Universität München
- Felix Pförtner: The first two authors contributed equally to this work
- Eva Briem: The first two authors contributed equally to this work
Protocol Citation: Felix Pförtner, Eva Briem, Wolfgang Enard, Daniel Richter 2025. prime-seq 2. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn97kpl5d/v1
Manuscript citation:
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: Working
We use this protocol and it's working
Created: December 02, 2024
Last Modified: August 20, 2025
Protocol Integer ID: 113399
Keywords: bulk RNA sequencing, RNA-seq, transcriptomics, SCRB-seq, prime-seq, efficient bulk rna, best bulk rna, seq protocol, increased final unique molecular identifier, sequencing cost, seq option, seq, final unique molecular identifier, optimized protocol, addressing dnase digestion, transcription, dnase digestion
Funders Acknowledgements:
DFG
Grant ID: 238187445
Disclaimer
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Abstract
We systematically optimized the powerful and cost-efficient bulk RNA-seq protocol prime-seq. Notable efficiency enhancements were achieved by addressing DNase digestion, reverse transcription, adapter ligation, and amplification.
The optimized protocol increased final unique molecular identifiers (UMIs) by 60 % at equal sequencing costs or, conversely, reduced sequencing costs by 38 % at equal counts. This further improves one of the most cost-efficient bulk RNA-seq protocols available, making it currently one of the best bulk RNA-seq options.
Guidelines
- All reagents and plastic-ware can be found in the 'Materials' section.
- Use only RNase free supplies and clean all surfaces and tools with RNase Away prior to working
- Make sure all steps involving cell lysate and RNA before reverse transcription are carried out swiftly and on ice.
- All primer sequences are listed below:
| A | B | C | D | E | F | |
| Oligo | Vendor | Purification | Working Conc. | Sequence ("*" = PTO bond) | Notes | |
| Barcoded Oligo-dT (E3V7NEXT) | Sigma | Cartridge | 10 µM | ACACTCTTTCCCTACACGACGCTCTTCCGATCT[12 bp BC]NNNNNNNNNNNNNNNVTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN | ||
| Template Switching Oligo (TSO) (E5V7NEXT) | Sigma | RNase-Free HPLC | 100 µM | Biotin-ACACTCTTTCCCTACACGACGCrGrGrG | ||
| Preamp Primer (SINGV6 PTO) | Sigma | Standard Desalting | 10 µM | Biotin-ACACTCTTTCCCTACACGAC*G*C | ||
| 3' enrichment primer (P5NEXTPT5) | Sigma | Standard Desalting | 5 µM | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT | ||
| i7 Index Primer PTO (Nextera) | IDT | Trugrade | 5 µM | CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGGAGATGTGTATAAGAGAC*A*G | ||
| i5 Index Primer PTO (TruSeq) | IDT | Trugrade | 5µM | AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGAT*C*T | ||
| blocked prime-seq Adapter AntiSense | IDT | Standard Desalting | 1.5 µM | Phos-CTGTCTCTTATACACATCTddC | Duplexed DNA | |
| blocked prime-seq Adapter Sense | IDT | Standard Desalting | 1.5 µM | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGT | Duplexed DNA |
Specific barcoded oligodT (E3V7NEXT) sequences:
E3V7_Set1.txt E3V7_Set2.txt Materials
MATERIALS
DNase I Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0303S
DNase I (RNase-free) - 1,000 unitsNew England BiolabsCatalog #M0303S
Deoxynucleotide Solution Mix - 40 umol of eachNew England BiolabsCatalog #N0447L
Exonuclease I (E.coli) - 3,000 unitsNew England BiolabsCatalog #M0293S
Quant-it™ PicoGreen® dsDNA Assay KitLife TechnologiesCatalog #P7589
ß -mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M3148
QuantiFluor(R) RNA SystemPromegaCatalog #E3310
Proteinase K solution, 20 mg ml − 1AmbionCatalog #AM2546
5 M Sodium chloride (NaCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5150-1L
Agilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
Buffer RLT PlusQiagenCatalog #1053393
Maxima H Minus Reverse Transcriptase (200 U/uL)Thermo Fisher ScientificCatalog #EP0752
NEBNext Ultra II FS DNA Library Prep with Sample Purification Beads - 24 rxnsNew England BiolabsCatalog #E6177S
EDTAMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7889
Ethanol absoluteCarl RothCatalog #9065.4
IgepalMerck MilliporeSigma (Sigma-Aldrich)Catalog #I8896
KAPA HiFi 2x RMKapa BiosystemsCatalog #KR0370
Poly(ethylene glycol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #89510
UltraPure DNase/RNase Free Distilled WaterCatalog #10977-049
Trizma hydrochloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #T2694
Aluminium seals for cold storageCatalog #391-1275
Filter tips 96 low retention 10 uLCatalog #771265
PCR SealsThermo ScientificCatalog #AB0558
twin.tec 96-well DNA LoBind PlatesEppendorfCatalog #0030129504
Sera-Mag Speed BeadsGE HealthcareCatalog #65152105050250
Sodium AzideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2002-100G
Troubleshooting
Safety warnings
Please follow all Manufacturer safety warnings and recommendations.
Before start
Wipe bench surfaces with RNAse Away and keep working environment clean.
Preparation
12m
Clean all surfaces and pipettes with RNase Away
5m
Thaw frozen buffers and primers on ice
10m
Prepare 80% EtOH (approximately 45 mL for 96 samples)
2m
When running the protocol for the first time prepare Cleanup Beads (see end of the protocol)!
45m
prime-seq can be used on lysate or extracted RNA. It is essential, however, that the samples either have the same input or that they are normalized after the RNA is extracted, otherwise sequencing depth per sample will be impacted. Based on your starting material, please follow one of the following cases:
Lysate (similar input), Direct Lysis107 steps
Follow this case if you are testing samples that have similar input (i.e. the expected RNA amount is the same between samples). The steps here will guide you in digesting residual proteins in your samples, extracting the RNA, digesting DNA, preparing RNA-seq libraries, and finally sequencing.
Example: investigating the genotype effect on transcription in 5,000 neurons
First Time Setup
When running the direct lysis protocol for the first time, prepare Bead Binding Buffer (see end of the protocol)!
Sample Collection
Prepare Lysis Buffer according to the number of samples.
| Reagent | Well | Plate | |
| RLT Plus Buffer | 99 µL | 10.89 mL | |
| β-mercaptoethanol | 1 µL | 110 µL | |
| Total | 100 µL | 11 mL |
Note
If sample volume exceeds 25 % of total lysate, use 2x TCL buffer
(Qiagen, #1070498) + 1 % β-mercaptoethanol
2m
Add100 µL of Lysis Buffer to each well of a semi-skirted 96-well PCR plate
1m
Add cells or tissue to wells
Note
Cells
Minimum: 100 cells, Optimum: 10,000 cells
Make sure that the same number of cells are used for each sample. Large differences between cells will impact distribution of sequencing reads and can potentially affect normalization.
Note
Tissue
If samples are difficult to lyse they should be homogenized using a tissue homogenizer.
Tissue should be a relatively small and not exceed more than 1000 ng of RNA. Tissue samples should be normalized by weight and be the same type of tissue.
Large differences between tissue samples will impact distribution of sequencing reads and can potentially affect normalization.
If you are unsure if the samples will contain the same amount of RNA, it is best to switch to the "Lysate (variable)" case in Step 13.
Transfer 50 µL of lysate to a new plate, return one plate immediately to -80 C freezer to save as a backup
Note
Conversely, one can prepare two plates during sorting with 50 µL of lysis buffer.
1m
Proteinase K Digest
30m
Prepare Proteinase K Mix
| A | B | C | |
| Reagent | Well | Plate | |
| EDTA (50mM) | 0.5 µL | 55 µL | |
| H2O | 0.5 µL | 55 µL | |
| Proteinase K (20mg/mL) | 1 µL | 110 µL | |
| Total | 2 µL | 220 µL |
Add 2 µL of Proteinase K Mix and mix by pipetting
1m
Incubate for 00:15:00 at 50 °C and then heat inactivate the Proteinase K for 00:10:00 at 75 °C
25m
Bead Clean Up
20m
Mix each bulk sample (50 µL per well) with 100 µL of Cleanup Beads (22% PEG)
1m
Incubate for 00:05:00 at 20 °C (Room Temp)
Note
While binding, prepare DNase I Mix (Step 28)
5m
Place on magnet stand until clear (~3 min) and then discard supernatant
3m
Wash with 100 µL of 80% EtOH while the plate is on the magnet. Discard the supernatant
Note
After adding EtOH, incubate for 30 s so that all beads are bound to magnet.
2m
Repeat wash step once more
2m
Air dry beads for 00:03:00
Note
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
3m
DNAse I Digest
1m
Add 10 µL H2O and resuspend beads by vortexing vigorously
Note
If you encounter bead clumping at this step, try to resuspend the beads by vigorously pipetting the samples. We have generated high quality prime-seq libraries despite heavy clumping.
2m
Prepare DNase I Mix
| A | B | C | |
| Reagent | Well | Plate | |
| DNase I | 1 µL | 110 µL | |
| DNase I Buffer (10x) | 2 µL | 220 µL | |
| H2O | 7 µL | 770 µL | |
| Total | 10 µL | 1.1 mL |
3m
Add 10 µL of DNase I Mix and mix by pipetting
2m
Incubate DNase I Mix and beads for 00:10:00 at 20 °C (Room Temp)
10m
Heat inactivate the DNase I by adding 1 µL of EDTA (50 mM) and 20 µL of Bead Binding Buffer (2X) and incubating for 00:05:00 at 65 °C
6m
Place plate on magnet stand until clear (~3 min) and discard the supernatant.
3m
Wash with 100 µL of 80% EtOH while the plate is on the magnet. Discard the supernatant
2m
Repeat wash step once more
2m
Air dry beads for 00:05:00
Note
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
Note
While drying, prepare Reverse Transcription Mix.
5m
Reverse Transcription
5m
Prepare Reverse Transcription Mix
| A | B | C | |
| Reagent | Well | Plate | |
| Maxima H Minus RT | 0.15 µL | 16.5 µL | |
| Maxima RT Buffer (5x) | 2 µL | 220 µL | |
| dNTPs (25 mM) | 0.4 µL | 44 µL | |
| TSO (E5V7NEXT) (100 µM) | 0.1 µL | 11 µL | |
| UltraPure Water | 2.35 µL | 258.5 µL | |
| Total | 5 µL | 550 µL |
5m
Add 4 µL H2O to the beads
Note
The 4 µL of water can be combined with the Reverse Transcription Mix by increasing the water in Row 6 from 2.35 µL to 6.35 µL.
If working with many samples, or if using a stepper pipette or robot, we find that it is better to add some water separately to prevent the beads from drying too much.
1m
Add 5 µL Reverse Transcription Mix
1m
Add 1 µL of Barcoded oligodT (E3V7NEXT) (10 µM) per well
2m
Incubate for 01:30:00 at 42 °C
1h 30m
cDNA Pooling & Purification
5m
Place the plate on a magnet
3m
Pool the supernatant of all wells into a 2 mL tube
10m
Add 10 µL of Cleanup Beads (22% PEG) per sample for a 1:1 ratio (e.g. 240 µL for 24 samples)
Note
The EDTA in the Cleanup Beads (22% PEG) will inactivate the RT.
5m
Incubate for 00:05:00 at Room temperature to allow binding of the cDNA onto beads
5m
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
3m
Wash with 1 mL of 80% EtOH while the tube is on the magnet, discard the supernatant
Note
Volume of EtOH should be adjusted depending on the number of samples. More samples will require more EtOH to cover the beads completely.
1m
Repeat wash step once more
1m
Air dry beads for 00:05:00
Note
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
5m
Elute the beads in 17 µL of UltraPure Water
1m
Incubate for 00:05:00 at RT and transfer to a new PCR tube or plate
5m
Exonuclease I Treatment
35m
Add 2 µL of ExoI Buffer (10x) and 1 µL of Exonuclease I. Incubate as follows:
| Step | Temperature | Time | |
| Incubation | 37 C | 20 min | |
| Heat Inactivation | 80 C | 10 min | |
| Storage | 4 C | ∞ |
35m
Mix each sample (20 µL per well) with 16 µL of Cleanup Beads (22% PEG) for a 1:0.8 ratio
1m
Incubate for 00:05:00 at Room temperature to allow binding of the cDNA onto beads
5m
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
3m
Wash with 50 µL of 80% EtOH while the tube is on the magnet, discard the supernatant
1m
Repeat wash step once more
1m
Air dry beads for 00:05:00
Note
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
5m
Elute the beads in 20 µL of UltraPure Water
1m
Incubate for 00:05:00 at RT and transfer to a new PCR tube or plate
5m
Full length cDNA Amplification
1m
Prepare Pre Amplification Mix
| A | B | |
| Reagent | 1x | |
| KAPA HiFi 2x RM | 25 µL | |
| Pre-amp Primer (SINGV6-PTO) (10 µM) | 3 µL | |
| UltraPure Water | 2 µL | |
| Total | 30 µL |
1m
Add 30 µL Pre Amplification Mix to sample
1m
Incubate the Pre Amplification PCR as follows:
| Step | Temperature | Time | Cycles | |
| Initial Denaturation | 98 C | 3 min | 1 cycle | |
| Denaturation | 98 C | 15 sec | 10 cycles* | |
| Annealing | 65 C | 30 sec | ||
| Elongation | 72 C | 4 min | ||
| Final Elongation | 72 C | 10 min | 1 cycle | |
| Storage | 4 C | ∞ |
Note
Adjust the number of cycles based on input (sample number, cell number, or concentration).
As a rule of thumb we assume big cells like embryonic stem cells to contain 10 pg of total RNA and small cells like T-cells ~ 1-2 pg
As a general guide we recommend:
| Total RNA Input | Cycles | |
| 10 ng | 16 | |
| 50 ng | 14 | |
| 100 ng | 12 | |
| 500 ng | 10 | |
| 1000 ng | 9 |
1h 30m
cDNA Bead Purification
1m
Mix sample with 40 µL Clean Up Beads (22% PEG) for a ratio of 1:0.8
1m
Incubate for 00:05:00 at 20 °C (Room Temp)
5m
Place the tube on the magnet stand until clear (~3 min) and discard supernatant
3m
Wash with 100 µL of 80% EtOH while the tube is on the magnet, discard the supernatant
1m
Repeat wash step once more
1m
Air dry beads for 00:05:00
Note
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
5m
Elute cDNA in 10 µL Buffer EB
1m
Incubate for 00:05:00 at RT and transfer to a new PCR tube or plate
Note
Stopping Point. Samples can be safely stored at -20 °C and protocol can be continued at a later date.
5m
cDNA Quantification and Quality Check
45m
Quantify the cDNA using the QuantiFluor dsDNA System or equivalent Qubit following the manufacturer's protocol. Use 1 μl of clean cDNA for quantification.
10m
Quality check the cDNA using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.
Note
Passing the cDNA quality check does not guarantee that the data will be of high quality, however, if the cDNA fails the quality check it will usually not yield good libraries and will therefore generate lower quality data.
Expected result
45m
Library Preparation
Note
Before starting, read the library preparation section carefully as there are a few steps that are very time sensitive.
Prepare Fragmentation Mix
| A | B | |
| Reagent | 1x | |
| Ultra II FS Reaction Buffer | 1.4 µL | |
| Ultra II FS Enzyme Mix | 0.4 µL | |
| cDNA (4-8 ng/µL) | 2.5 µL | |
| Buffer TE | 1.7 µL | |
| Total | 6 µL |
Note
Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.
Note
Vortex the Ultra II FS Enzyme Mix for 5-8 seconds prior to use for optimal performance.
1m
Vortex the Fragmentation Mix for00:00:05 and immediately proceed to the next step
10s
Incubate the Fragmentation reaction as follows:
| A | B | C | |
| Step | Temperature | Time | |
| Pre-Cool | 4 C | ∞ | |
| Fragmentation | 37 C | 5 min* | |
| A Tailing and Phosphorylation | 65 C | 30 min | |
| Storage | 4 C | ∞ |
Note
*Fragmentation time needs to be adjusted for very low (<5ng)/ high (>50ng) cDNA amounts.
Note
Set heated lid to 75° C. Make sure the lid is at the correct temperature before you start the reaction.
Skip the first incubation step once you have added your samples.
40m
Adapter Ligation
20m
Prepare Adapter Ligation Mix
| A | B | |
| Reagent | 1x | |
| NEBNext Ultra II Ligation Master Mix | 6 µL | |
| NEBNext Ligation Enhancer | 0.2 µL | |
| blocked prime-seq Adapter (1.5 µM) | 0.5 µL | |
| Total | 6.7 µL |
1m
Add 6.7 µL Adapter Ligation Mix to each replicate
1m
Incubate for 00:15:00 at 20 °C
Note
Turn off heated lid
15m
Add 37.3 µL Buffer EB to Samples
1m
Mix Sample with 26 µL SPRI select beads
Note
We use SPRI Select Beads here instead of our home made 22% Clean Up beads for their guaranteed QCed size selection properties.
Note
The volume of SPRI select beads used during library size selection can be adjusted based on desired library size. Optimization for your samples may be required.
1m
Incubate for 00:05:00 at 20 °C (Room Temp)
5m
Place the plate on the magnet stand until clear and transfer76 µL supernatant to clean well.
Note
Be careful not to discard! This is your sample!
3m
Mix supernatant with 10 µL SPRI select beads
Note
The volume of SPRI select beads used during library size selection can be adjusted based on desired library size. Optimization for your samples may be required.
1m
Incubate for 00:05:00 at 20 °C (Room Temp)
5m
Place the plate on the magnet stand until clear and discard supernatant
3m
Wash with 150 µL of 80% EtOH while the plate is on the magnet, discard the supernatant
1m
Repeat wash step once more
1m
Air dry beads for 00:05:00
Note
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
5m
Elute samples in 11 µL 0.1X TE (dilute 1X TE Buffer 1:10 in water) for 00:05:00
5m
Library PCR
45m
Transfer 10.5 µL of samples to clean wells
Add 1 µL of Index Primer (Nextera i7, 5 uM) to each well
Note
This is the unique index that will be used for demultiplexing libraries.
Add 1 µL of Index Primer (TruSeq i5, 5 uM) to each well
Note
Alternatively the universal primer P5NEXTPT5 can be used in case the second index will not be sequenced.
Prepare Library PCR Mix by adding 12.5 µL of Q5 Master Mix
Note
Although scaled down, there will not be sufficient Q5 Master Mix (M0544L) in the kit. This item will have to be ordered separately.
Incubate the Library PCR reaction as follows:
| A | B | C | D | |
| Step | Temperature | Time | Cycles | |
| Initial Denaturation | 98 C | 30 sec | 1 cycle | |
| Denaturation | 98 C | 10 sec | 10 cycles* | |
| Annealing/Elongation | 72 C | 1 min 15 sec | ||
| Final Elongation | 72 C | 5 min | 1 cycle | |
| Storage | 4 C | ∞ |
Note
* Adjust the number of cycles based on cDNA input.
As a general guide we recommend:
| cDNA Input | Cycles | |
| 20 ng | 10 | |
| 10 ng | 11 | |
| 5 ng | 12 |
Double Size Selection
25m
Add 25 µL Buffer EB to Index PCR
Mix Index PCR with 26 µL SPRI select beads
Note
We use SPRI Select Beads here instead of our home made 22% Clean Up beads for their guaranteed QCed size selection properties.
Note
The volume of SPRI select beads used during library size selection can be adjusted based on desired library size. Optimization for your samples may be required.
Incubate for 00:05:00 at 20 °C (Room Temp)
Place the plate on the magnet stand until clear and transfer 76 µL supernatant to clean well.
Note
Be careful not to discard! This is your library.
Mix supernatant with 10 µL SPRI select beads
Note
The volume of SPRI select beads used during library size selection can be adjusted based on desired library size. Optimization for your samples may be required.
Incubate for 00:05:00 at 20 °C (Room Temp)
Place the plate on the magnet stand until clear and discard supernatant.
Wash with 150 µL of 80% EtOH while the plate is on the magnet, discard the supernatant
Repeat wash step once more
Air dry beads for 00:05:00
Note
Depending on temperature and humidity, the beads may dry faster. Therefore it is important to regularly check the beads and avoid over-drying.
Elute in 15 µL Buffer EB.
Incubate for 00:05:00 and then place on magnet until clear. Transfer eluted library to new well.
Note
Stopping point. The libraries can be safely stored at -20 °C until they will be QCed and sequenced.
QC and quantification
45m
Quantify and quality control the library using the Agilent 2100 Bioanalyzer with High Sensitivity DNA Analysis Kits.
Note
Bulk libraries often yield high concentrations, which should be diluted to get accurate molarity measurements on the Bioanalyzer. Ideally, do not load more than 2 ng onto the chip.
Expected result
Libraries will typically exceed 1-5 ng/μl concentration
Sequencing
1m
Samples should be submitted according to your Sequencing Facility specifications. prime-seq is compatible with Illumina Sequencing.
At least 8 cycles are required for the Index Read (i7) and 28 cycles for the Read 1 (BC+UMI). Dual index sequencing can be done when using patterned flowcells. Read 2 (DNA) should be adjusted based on the quality of the genome being mapped to, but for human and mouse 50 cycles is sufficient.
Some potential sequencing options:
| A | B | C | D | E | F | |
| Sequencer | Read 1 | Read 2 | Index Read (i7) | Index Read (i5) | Kit | |
| NovaSeq | 28 | 94 | 8 | 8 | NovaSeq SP v1.5 100 cycle | |
| NextSeq 500/550 | 28 | 56 | 8 | 0 | NextSeq 500/550 HiOut v3 75 cycle | |
| NextSeq 1000/2000 | 28 | 94 | 8 | 8 | NextSeq 1000/2000 P2 100 cycle | |
| NextSeq 2000 | 28 | 52 | 8 | 0 | NextSeq 2000 P3 50 cycles | |
| HiSeq | 28 | 114 | 8 | 0 | HiSeq 3000/4000 150 cycles |
Prepare Cleanup Beads (22% PEG)
10m
Prepare PEG Solution (22%) by adding all ingredients to a 50 mL falcon tube
| Reagent | Amount | |
| PEG 8000 | 11 g | |
| NaCl (5M) | 10 mL | |
| Tris-HCl (1M, pH 8.0) | 500 μL | |
| EDTA (0.5M) | 100 μL | |
| IGEPAL (10% solution) | 50 μL | |
| Sodium Azide (10% solution) | 250 μL | |
| UltraPure Water | up to 49 mL | |
| Total | 49 mL |
Note
Do not add the total amount of water until after PEG is completely solubilized
10m
Incubate at 40 °C and vortex regularly until PEG is completely dissolved
10m
Resuspend Sera-Mag Speed Beads carefully and pipette 1000 µL of bead suspension into a 1.5 mL tube
1m
Place on magnet stand and remove storage buffer
1m
Add 1000 µL of TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads
30s
Place on magnet stand and remove supernatant
30s
Repeat wash step one more time
1m
Add 900 µL TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and resuspend beads
30s
Add the washed Sera-Mag Speed Beads to the PEG Solution (22%) and mix well
Note
The final Cleanup Beads (22% PEG) can be aliquoted and stored at 4 °C for up to six months
1m 30s
Prepare Bead Binding Buffer
10m
Prepare Bead Binding Buffer (2x)
| Reagent | ||
| PEG 8000 | 1.1 g | |
| NaCl (5 M) | 1 mL | |
| Tris-HCl (1 M, pH 8.0) | 50 µL | |
| Igepal (10% solution) | 5 µL | |
| Sodium Azide (10% solution) | 25 µL | |
| H2O | to 5 mL | |
| Total | 5 ml |
Note
The Bead Binding Buffer (2x) can be stored at Room temperature for up to six months.
10m
Protocol references
Janjic, A., Wange, L.E., Bagnoli, J.W. et al. Prime-seq, efficient and powerful bulk RNA sequencing.
Genome Biol 23, 88 (2022). https://doi.org/10.1186/s13059-022-02660-8