Sep 04, 2020
Plate based scRNA-seq Illumina library construction
- DNA Pipelines R&D1,
- Peter Ellis1,
- Lesley Shirley1
- 1Wellcome Sanger Institute
Protocol Citation: DNA Pipelines R&D, Peter Ellis, Lesley Shirley 2020. Plate based scRNA-seq Illumina library construction. protocols.io https://dx.doi.org/10.17504/protocols.io.bi4fkgtn
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: July 29, 2020
Last Modified: September 04, 2020
Protocol Integer ID: 39783
Abstract
This SOP describes the procedure for plate based scRNA-seq performed with a commercial available kit from New England BioLabs. Following library construction, samples are pooled in equivolume and quantified, prior to sequencing on the Illumina HiSeq 4000 platform.
Guidelines
It is vital all steps prior to cDNA amplification are performed in a designated RNase free and pre-cDNA amplification laboratory.
Note
Throughout the protocol we have indicated the liquid handling in use at Sanger for specific parts of the process. However, these steps can be performed on alternative liquid handlers.
Materials
MATERIALS
KAPA HiFi HotStart ReadyMixPCR KitKapa BiosystemsCatalog #KK2602
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina - 96 rxns New England BiolabsCatalog #E6420L
AccuClear® Ultra High Sensitivity dsDNA Quantitation Kit with DNA StandardsBiotiumCatalog ##31028
Bioanalyzer chips and reagents (DNA 1000)Agilent TechnologiesCatalog #5067-1504
AMPure XP BeadsBeckman CoulterCatalog #A63882
STEP MATERIALS
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina - 96 rxns New England BiolabsCatalog #E6420L
AccuClear® Ultra High Sensitivity dsDNA Quantitation Kit with DNA StandardsBiotiumCatalog ##31028
2x Kapa HiFi Hotstart Readymix Kapa BiosystemsCatalog #KK2602
Bioanalyzer chips and reagents (DNA 1000)Agilent TechnologiesCatalog #5067-1504
AMPure XP BeadsBeckman CoulterCatalog #A63882
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina - 96 rxns New England BiolabsCatalog #E6420L
AMPure XP BeadsBeckman CoulterCatalog #A63882
AMPure XP BeadsBeckman CoulterCatalog #A63882
AMPure XP BeadsBeckman CoulterCatalog #A63882
Protocol materials
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina - 96 rxns New England BiolabsCatalog #E6420L
2x Kapa HiFi Hotstart Readymix Kapa BiosystemsCatalog #KK2602
Bioanalyzer chips and reagents (DNA 1000)Agilent TechnologiesCatalog #5067-1504
KAPA HiFi HotStart ReadyMixPCR KitKapa BiosystemsCatalog #KK2602
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina - 96 rxns New England BiolabsCatalog #E6420L
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina - 96 rxns New England BiolabsCatalog #E6420L
AMPure XP BeadsBeckman CoulterCatalog #A63882
AccuClear® Ultra High Sensitivity dsDNA Quantitation Kit with DNA StandardsBiotiumCatalog ##31028
AMPure XP BeadsBeckman CoulterCatalog #A63882
AMPure XP BeadsBeckman CoulterCatalog #A63882
AMPure XP BeadsBeckman CoulterCatalog #A63882
Bioanalyzer chips and reagents (DNA 1000)Agilent TechnologiesCatalog #5067-1504
AccuClear® Ultra High Sensitivity dsDNA Quantitation Kit with DNA StandardsBiotiumCatalog ##31028
AMPure XP BeadsBeckman CoulterCatalog #A63882
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina - 96 rxns New England BiolabsCatalog #E6420L
AMPure XP BeadsBeckman CoulterCatalog #A63882
AccuClear® Ultra High Sensitivity dsDNA Quantitation Kit with DNA StandardsBiotiumCatalog ##31028
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina - 96 rxns New England BiolabsCatalog #E6420L
AMPure XP BeadsBeckman CoulterCatalog #A63882
AMPure XP BeadsBeckman CoulterCatalog #A63882
2x Kapa HiFi Hotstart Readymix Kapa BiosystemsCatalog #KK2602
AMPure XP BeadsBeckman CoulterCatalog #A63882
Bioanalyzer chips and reagents (DNA 1000)Agilent TechnologiesCatalog #5067-1504
Preparation of lysis buffer plates and FACS
Preparation of lysis buffer plates and FACS
Important! This step must be performed in a designated RNAse free and pre-cDNA amplification area, keeping reagents chilled at all times
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina - 96 rxns New England BiolabsCatalog #E6420L
Prepare the cell lysis buffer, which will provide sufficient volume for one 96-well plate On ice
| Reagent | Volume (µl) | |
| NEBNext Cell Lysis Buffer (10x) | 24 | |
| Murine RNase Inhibitor | 12 | |
| Nuclease-Free Water | 204 | |
| Total | 240 |
Mix well by pipetting.
Use the Formulatrix Mantis microfluidic liquid handler to dispense 2 µL of lysis buffer into a 96-well PCR plate.
Note
If required add a diluted stock (1/500,000) of ERCCs into the lysis buffer.
Seal dispensed plates, centrifuge immediately 1000 x g, 4°C, 00:01:00 and keep chilled on ice.
PAUSE POINT Lysis buffer plates can be stored at -80 °C prior to cell sorting. Plates can be stored for < 6 months.
Note
When FACS sorting, take care of plate calibration/priming prior to single-cell deposition. If many plates are deposited in parallel, repeat the calibration/priming at least every 8 plates.
Defrost lysis buffer plates prior to cell sorting, centrifuge 1000 x g, 4°C, 00:01:00 and keep chilled on ice.
After FACS sorting, seal and centrifuge plates immediately 1000 x g, 4°C, 00:01:00 and keep chilled on ice.
PAUSE POINT Plates of sorted cells can be stored at -80 °C for < 6 months. the quality of the data depends on the cell type and duration of storage.
Primer Annealing for first-strand synthesis
Primer Annealing for first-strand synthesis
Prepare the primer annealing mix, which will provide sufficient volume for one 96-well plate On ice
| Reagent | Volume (µl) | |
| NEBNext Single Cell RT Primer Mix | 50 | |
| Nuclease-Free Water | 250 | |
| Total | 300 |
Mix well by pipetting.
The Agilent Bravo with 96 ST head will combine 1.6 µL of primer annealing mix with 2 µL of lysed cells and mix by pipetting.
Seal and transfer the plate to a thermocycler with the heated lid set to 100 °C and run the following program:
| Temperature | Time | |
| 70ºC | 5 minutes | |
| 4ºC | ∞ |
Reverse transcription (RT) and template switching
Reverse transcription (RT) and template switching
Prepare the RT mix, which will provide sufficient volume for one 96-well plate On ice
| Reagent | Volume (µl) | |
| NEBNext Single Cell RT Buffer | 250 | |
| NEBNext Template Switching Oligo | 50 | |
| NEBNext Single Cell RT Enzyme | 150 | |
| Nuclease-Free Water | 100 | |
| Total | 550 |
Mix well by pipetting.
The Bravo will add 4.4 µL of RT mix to each sample and mix by pipetting.
Seal and transfer the plate to a thermocycler with the heated lid set to 100 °C and run the following program:
| Temperature | Time | |
| 42ºC | 90 minutes | |
| 70ºC | 10 minutes | |
| 4ºC | ∞ |
Prepare the cDNA amplification mix, which will provide sufficient volume for one 96-well plate On ice
| Reagent | Volume (µl) | |
| NEBNext Single Cell cDNA PCR Master Mix | 2500 | |
| NEBNext Single Cell cDNA Primer Mix | 100 | |
| Nuclease-Free Water | 1400 | |
| Total | 4000 |
Mix well by pipetting.
The Bravo will add 32 µL of cDNA amplification mix to each sample and mix by pipetting.
Seal and transfer the plate to a thermocycler with the heated lid set to 100 °C and run the following program:
| Temperature | Time | Cycles | |
| 98ºC | 45 seconds | 1 | |
| 98ºC | 10 seconds | 16-25 depending on cell type | |
| 62ºC | 15 seconds | ||
| 72ºC | 3 minutes | ||
| 72ºC | 5 minutes | 1 | |
| 4ºC | ∞ | 1 |
Purification of amplified cDNA
Purification of amplified cDNA
AMPure XP BeadsBeckman CoulterCatalog #A63882
Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.
Centrifuged amplified cDNA plate 1000 x g, 00:01:00
Use the Agilent Bravo with a 96 LT multichannel head to perform the following steps:
Add 0.6 X volume of SPRI beads per sample (24 µL SPRI : 40 µL amplified cDNA), mix well by pipetting.
Incubate for 00:05:00 at Room temperature
Transfer the plate to the magnet, allow 00:02:00 for the beads to settle.
Carefully remove and discard the supernatant without disturbing the bead pellet.
Wash the beads with 180 µL 80% freshly prepared ethanol for 00:00:30 remove ethanol and discard.
Repeat ethanol wash.
Allow beads to dry 00:05:00
Remove the plate from the magnet, add 50 µL nuclease-free water and resuspend by mixing well.
Incubate for 00:02:00 at Room temperature
Transfer the plate to the magnet, allow 00:05:00 for the beads to settle.
Transfer supernatant into a new 96-well PCR plate, taking care not to disturb the pellet.
Quality control of amplified cDNA
Quality control of amplified cDNA
Note
Purified amplified cDNA is quantified with a fluorescence based assay. We use the AccuClear Ultra High Sensitivity dsDNA Quantitation kit with 7 DNA standards (Biotium) according to manufacturer's instructions.
To streamline the workflow, we do not normalise sample input for library preparation. Instead, we calculate an average concentration and transfer a fixed volume such that 5-25 ng of each successfully amplified cDNA sample enters library preparation.
AccuClear® Ultra High Sensitivity dsDNA Quantitation Kit with DNA StandardsBiotiumCatalog ##31028
Pipette 20 µL of each DNA standard into wells A1 - G1 of a 96-well PCR plate. Add nuclease-free water to H1.
Dilute the AccuClear dye (100X) to working concentration by mixing 300 µL dye with 30 mL AccuClear buffer in a 50 ml Falcon. Mix thoroughly by vortexing and transfer to a 384-well reservoir.
Use the SPT Labtech Mosquito LV to stamp 200 nl of amplified cDNA and 1 µL of known standards in triplicate into a 384-well assay plate. Immediately proceed to the next step.
Use the Agilent Bravo with a 384ST multichannel head to add 50 µL 1 X AccuClear dye from the reservoir to the assay plate, mix thoroughly by pipetting.
Measure fluorescence values on a BMG FLUOstar Omega plate reader calibrated for use with AccuClear dye.
Confirm known standards are performing as expected.
Dilute any samples >125 ng/µl with nuclease free water so they are in the range of 10 - 125 ng/µl and repeat quantitation.
Note
We use 5X the volume of standard vs sample in our assay setup, which should allow a quantitative range of 0.15 ng/µl - 125 ng/µl. This deviates from the standard kit SOP which has a stated range of 0.03 ng/µl - 25 ng/µl.
Taking an average across the plate. Transfer ~ 10 ng of cDNA into a new 96-well PCR plate for sequencing library preparation.
PAUSE POINT Purified amplified cDNA can be stored at -20 °C for several weeks prior to library preparation.
Illumina sequencing library preparation
Illumina sequencing library preparation
Note
We use the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina for library preparation, which we have automated on the Agilent Bravo NGS platform with some modifications. We use a custom adapter set, however any TruSeq adapters are suitable.
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina - 96 rxns New England BiolabsCatalog #E6420L
AMPure XP BeadsBeckman CoulterCatalog #A63882
Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.
Centrifuged plate containing ~ 10 ng of purified cDNA 1000 x g, 00:01:00
Use the Agilent Bravo with a 96 LT multichannel head to perform the following steps:
Add 0.9 X volume of SPRI beads per sample, mix well by pipetting.
Incubate for 00:05:00 at Room temperature
Transfer the plate to the magnet, allow 00:02:00 for the beads to settle.
Carefully remove and discard the supernatant without disturbing the bead pellet.
Wash the beads with 180 µL 80% freshly prepared ethanol for 00:00:30 remove ethanol and discard.
Repeat ethanol wash.
Allow beads to dry 00:05:00
Remove the plate from the magnet, add 13 µL TE pH 8.0 and resuspend by mixing well.
Incubate for 00:02:00 at Room temperature
Transfer the plate to the magnet, allow 00:05:00 for the beads to settle.
Transfer 12.4 µL into a new 96-well PCR plate, taking care not to disturb the pellet.
Prepare fragmentation/end prep mix, which will provide sufficient volume for one 96-well plate On ice
| Reagent | Volume (µl) | |
| NEBNext Ultra II FS Reaction Buffer | 336 | |
| NEBNext Ultra II FS Reaction Enzyme | 96 | |
| Total | 432 |
Mix well by pipetting.
The Bravo will add 3.6 µL of fragmentation/end prep mix to each sample and mix by pipetting.
Seal and transfer the plate to a thermocycler with the heated lid set to 100 °C and run the following program:
| Temperature | Time | |
| 72ºC | 15 minutes | |
| 65ºC | 30 minutes | |
| 4ºC | ∞ |
Prepare adapter ligation mix, which will provide sufficient volume for one 96-well plate On ice
| Reagent | Volume (µl) | |
| NEBNext Ultra II Ligation Master Mix | 1440 | |
| NEBNext Ultra II Ligation Enhancer | 48 | |
| TruSeq Duplexed Adapter (100 µM) | 12 | |
| Nuclease-Free Water | 108 | |
| Total | 1608 |
Mix well by pipetting.
The Bravo will add 13.4 µL of ligation mix to each sample and mix by pipetting.
The plate is incubated on deck at 20 °C for 00:15:00 , however this step may also be performed on a thermocycler.
Note
We use alternative TruSeq compatible adapters, which do not require the USER enzyme incubation step. If using NEBNext adapters, follow the steps in the NEB protocol to add USER enzyme to the ligation reaction.
AMPure XP BeadsBeckman CoulterCatalog #A63882
Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.
Add 0.7 X volume of SPRI beads per sample (20 µL SPRI : 29.4 µL amplified cDNA), mix well by pipetting.
Incubate for 00:05:00 at Room temperature
Transfer the plate to the magnet, allow 00:02:00 for the beads to settle.
Carefully remove and discard the supernatant without disturbing the bead pellet.
Wash the beads with 180 µL 80% freshly prepared ethanol for 00:00:30 remove ethanol and discard.
Repeat ethanol wash.
Allow beads to dry 00:05:00
Remove the plate from the magnet, add 25 µL nuclease-free water and resuspend by mixing well.
Incubate for 00:02:00 at Room temperature
Transfer the plate to the magnet, allow 00:05:00 for the beads to settle.
Transfer supernatant into a new 96-well PCR plate, taking care not to disturb the pellet.
Note
We use KAPA HiFi HotStart ReadyMix and unique dual indexed (UDI) tag plates for library PCR.
Note: this deviates from the standard NEB protocol which uses NEBNext Ultra II Q5 Master Mix and different cycling conditions.
2x Kapa HiFi Hotstart Readymix Kapa BiosystemsCatalog #KK2602
Prepare PCR mix, which will provide sufficient volume for one 96-well plate On ice
| Reagent | Volume (µl) | |
| KAPA HiFi HotStart ReadyMix | 3000 | |
| Total | 3000 |
The Bravo will add 25 µL PCR mix and 25 µL sample into a lyophilised plate of UDIs and mix thoroughly by pipetting. The final concentration of each UDI in the PCR reaction is 2 µM.
Seal and transfer the plate to a thermocycler with the heated lid set to 100 °C and run the following program:
| Temperature | Time | Cycles | |
| 98ºC | 45 seconds | 1 | |
| 98ºC | 10 seconds | 8 | |
| 62ºC | 15 seconds | ||
| 72ºC | 3 minutes | ||
| 72ºC | 5 minutes | 1 | |
| 4ºC | ∞ | 1 |
PAUSE POINT amplified libraries can be stored at -20 °C for several weeks prior to library purification.
Pooling and purification of amplified libraries
Pooling and purification of amplified libraries
In a post-PCR lab, use the Hamilton STAR or Beckman NX-8 to combine 5 µL of each sample per plate to form an equivolume pool of 96 samples.
AMPure XP BeadsBeckman CoulterCatalog #A63882
Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.
Manually transfer 400 µL of the equivolume pool into a 1.5 ml Eppendorf tube
Add 0.95 X volume of SPRI beads (380 µL SPRI : 400 µL amplified libraries), mix well by pipetting.
Incubate for 00:05:00 at Room temperature
Transfer the tube to a magnet, allow 00:05:00 for the beads to form a pellet.
Carefully remove and discard the supernatant without disturbing the bead pellet.
Wash the beads with 180 µL 80% freshly prepared ethanol for 00:00:30 remove ethanol and discard.
Repeat ethanol wash.
Allow beads to dry 00:05:00
Remove the tube from the magnet, add 400 µL nuclease-free water and resuspend by mixing well.
Incubate for 00:02:00 at Room temperature
Transfer tube to magnet, allow 00:05:00 for the beads to form a pellet.
Transfer supernatant into a new tube, taking care not to disturb the pellet.
Quality control and normilisation of sequencing libraries
Quality control and normilisation of sequencing libraries
Note
Library sequencing pools are quantified on an Agilent Bioanalyzer. Pools are then diluted to 2.8 nM for sequencing.
Equipment
Bioanalyzer
NAME
Bioanalyzer
TYPE
Agilent
BRAND
G2991AA
SKU
LINK
Any bioanalyzer will suffice.
SPECIFICATIONS
Bioanalyzer chips and reagents (DNA 1000)Agilent TechnologiesCatalog #5067-1504
Run 1 µL of the library pool in triplicate on a Bioanalyzer using the DNA 1000 kit.
Taking an average of the readings add nuclease-free water to the library pool to produce a final concentration of 2.8 nM.
Sequencing
Sequencing
Note
We sequence samples on an Illumina HiSeq 4000 instrument (paired-end, 75-bp reads) according to the manufacturer's protocol. We typically aim for an average depth of 1 million reads per cell, plexing up to 384 samples per run.