TITLE: Plate based scRNA-seq Illumina library construction
AUTHORS: DNA Pipelines R&D, Peter Ellis, Lesley Shirley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [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
?. [Preparation of lysis buffer plates and FACS]
Prepare the cell lysis buffer, which will provide sufficient volume for one 96-well plate   AB1ReagentVolume (µl)2NEBNext Cell Lysis Buffer (10x)243Murine RNase Inhibitor124Nuclease-Free Water2045Total240Mix well by pipetting.
on ice
AB1ReagentVolume (µl)2NEBNext Cell Lysis Buffer (10x)243Murine RNase Inhibitor124Nuclease-Free Water2045Total240
?. [Preparation of lysis buffer plates and FACS]
Use the Formulatrix Mantis microfluidic liquid handler to dispense  of lysis buffer into a 96-well PCR plate.
2 µl
If required add a diluted stock (1/500,000) of ERCCs into the lysis buffer.
?. [Preparation of lysis buffer plates and FACS]
Seal dispensed plates, centrifuge immediately  and keep chilled on ice.
Centrifuge: 1000 34, 1 min, 4 10
?. [Preparation of lysis buffer plates and FACS]
PAUSE POINT Lysis buffer plates can be stored at  prior to cell sorting. Plates can be stored for
-80 °C
?. [Preparation of lysis buffer plates and FACS]
Defrost lysis buffer plates prior to cell sorting, centrifuge  and keep chilled on ice.
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.
Centrifuge: 1000 34, 1 min, 4 10
?. [Preparation of lysis buffer plates and FACS]
After FACS sorting, seal and centrifuge plates immediately  and keep chilled on ice.
Centrifuge: 1000 34, 1 min, 4 10
?. [Preparation of lysis buffer plates and FACS]
PAUSE POINT Plates of sorted cells can be stored at  for
-80 °C
?. [Primer Annealing for first-strand synthesis]
Prepare the primer annealing mix, which will provide sufficient volume for one 96-well plate   AB1ReagentVolume (µl)2NEBNext Single Cell RT Primer Mix503Nuclease-Free Water2504Total300Mix well by pipetting.
on ice
AB1ReagentVolume (µl)2NEBNext Single Cell RT Primer Mix503Nuclease-Free Water2504Total300
?. [Primer Annealing for first-strand synthesis]
The Agilent Bravo with 96 ST head will combine  of primer annealing mix with  of lysed cells and mix by pipetting.
1.6 µl
2 µl
?. [Primer Annealing for first-strand synthesis]
Seal and transfer the plate to a thermocycler with the heated lid set to  and run the following program:  AB1TemperatureTime270ºC5 minutes34ºC∞
100 °C
AB1TemperatureTime270ºC5 minutes34ºC∞
?. [Reverse transcription (RT) and template switching]
Prepare the RT mix, which will provide sufficient volume for one 96-well plate   AB1ReagentVolume (µl)2NEBNext Single Cell RT Buffer2503NEBNext Template Switching Oligo504NEBNext Single Cell RT Enzyme1505Nuclease-Free Water1006Total550Mix well by pipetting.
on ice
AB1ReagentVolume (µl)2NEBNext Single Cell RT Buffer2503NEBNext Template Switching Oligo504NEBNext Single Cell RT Enzyme1505Nuclease-Free Water1006Total550
?. [Reverse transcription (RT) and template switching]
Seal and transfer the plate to a thermocycler with the heated lid set to  and run the following program:  AB1TemperatureTime242ºC90 minutes370ºC10 minutes44ºC∞
100 °C
AB1TemperatureTime242ºC90 minutes370ºC10 minutes44ºC∞
?. [Reverse transcription (RT) and template switching]
Prepare the cDNA amplification mix, which will provide sufficient volume for one 96-well plate   AB1ReagentVolume (µl)2NEBNext Single Cell cDNA PCR Master Mix25003NEBNext Single Cell cDNA Primer Mix1004Nuclease-Free Water14005Total4000Mix well by pipetting.
on ice
AB1ReagentVolume (µl)2NEBNext Single Cell cDNA PCR Master Mix25003NEBNext Single Cell cDNA Primer Mix1004Nuclease-Free Water14005Total4000
?. [Reverse transcription (RT) and template switching]
The Bravo will add  of cDNA amplification mix to each sample and mix by pipetting.
32 µl
?. [Reverse transcription (RT) and template switching]
The Bravo will add  of RT mix to each sample and mix by pipetting.
4.4 µl
?. Seal and transfer the plate to a thermocycler with the heated lid set to  and run the following program:  ABC1TemperatureTimeCycles298ºC45 seconds1398ºC10 seconds16-25 depending on cell type462ºC15 seconds572ºC3 minutes672ºC5 minutes174ºC∞1
100 °C
ABC1TemperatureTimeCycles298ºC45 seconds1398ºC10 seconds16-25 depending on cell type462ºC15 seconds572ºC3 minutes672ºC5 minutes174ºC∞1
?. [Purification of amplified cDNA]
Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.
?. [Purification of amplified cDNA]
Centrifuged amplified cDNA plate
Centrifuge: 1000 34, 1 min
?. [Purification of amplified cDNA]
Use the Agilent Bravo with a 96 LT multichannel head to perform the following steps:
?. [Purification of amplified cDNA]
Add 0.6 X volume of SPRI beads per sample ( SPRI :  amplified cDNA), mix well by pipetting.
24 µl
40 µl
?. [Purification of amplified cDNA]
Incubate for  at
0 Room temperature
?. [Purification of amplified cDNA]
Transfer the plate to the magnet, allow  for the beads to settle.
?. [Purification of amplified cDNA]
Carefully remove and discard the supernatant without disturbing the bead pellet.
?. [Purification of amplified cDNA]
Wash the beads with  80% freshly prepared ethanol for  remove ethanol and discard.
180 µl
?. [Purification of amplified cDNA]
Repeat ethanol wash.
?. [Purification of amplified cDNA]
Allow beads to dry
?. [Purification of amplified cDNA]
Remove the plate from the magnet, add  nuclease-free water and resuspend by mixing well.
50 µl
?. [Purification of amplified cDNA]
Incubate for at
0 Room temperature
?. [Purification of amplified cDNA]
Transfer the plate to the magnet, allow  for the beads to settle.
?. [Purification of amplified cDNA]
Transfer supernatant into a new 96-well PCR plate, taking care not to disturb the pellet.
?. [Quality control of amplified cDNA]
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.
?. [Quality control of amplified cDNA]
Pipette  of each DNA standard into wells A1 - G1 of a 96-well PCR plate. Add nuclease-free water to H1.
20 µl
?. [Quality control of amplified cDNA]
Dilute the AccuClear dye (100X) to working concentration by mixing  dye with  AccuClear buffer in a 50 ml Falcon. Mix thoroughly by vortexing and transfer to a 384-well reservoir.
300 µl
30 mL
?. [Quality control of amplified cDNA]
Use the SPT Labtech Mosquito LV to stamp  of amplified cDNA and  of known standards in triplicate into a 384-well assay plate. Immediately proceed to the next step.
200
1 µl
?. [Quality control of amplified cDNA]
Use the Agilent Bravo with a 384ST multichannel head to add  1 X AccuClear dye from the reservoir to the assay plate, mix thoroughly by pipetting.
50 µl
?. [Quality control of amplified cDNA]
Measure fluorescence values on a BMG FLUOstar Omega plate reader calibrated for use with AccuClear dye.
?. [Quality control of amplified cDNA]
Confirm known standards are performing as expected.
?. [Quality control of amplified cDNA]
Taking an average across the plate. Transfer ~ 10 ng of cDNA into a new 96-well PCR plate for sequencing library preparation.
?. [Quality control of amplified cDNA]
Dilute any samples >125 ng/µl with nuclease free water so they are in the range of 10 - 125 ng/µl and repeat quantitation.
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.
?. [Illumina sequencing library preparation]
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.
?. [Illumina sequencing library preparation]
Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.
?. [Illumina sequencing library preparation]
Centrifuged plate containing ~ 10 ng of purified cDNA
Centrifuge: 1000 34, 1 min
?. [Illumina sequencing library preparation]
Use the Agilent Bravo with a 96 LT multichannel head to perform the following steps:
?. [Quality control of amplified cDNA]
PAUSE POINT Purified amplified cDNA can be stored at  for several weeks prior to library preparation.
-20 °C
?. [Illumina sequencing library preparation]
Add 0.9 X volume of SPRI beads per sample, mix well by pipetting.
?. [Illumina sequencing library preparation]
Incubate for  at
0 Room temperature
?. [Illumina sequencing library preparation]
Transfer the plate to the magnet, allow  for the beads to settle.
?. [Illumina sequencing library preparation]
Carefully remove and discard the supernatant without disturbing the bead pellet.
?. [Illumina sequencing library preparation]
Wash the beads with  80% freshly prepared ethanol for  remove ethanol and discard.
180 µl
?. [Illumina sequencing library preparation]
Repeat ethanol wash.
?. [Illumina sequencing library preparation]
Allow beads to dry
?. [Illumina sequencing library preparation]
Remove the plate from the magnet, add  TE pH 8.0 and resuspend by mixing well.
13 µl
?. [Illumina sequencing library preparation]
Incubate for at
0 Room temperature
?. [Illumina sequencing library preparation]
Transfer the plate to the magnet, allow  for the beads to settle.
?. [Illumina sequencing library preparation]
Transfer   into a new 96-well PCR plate, taking care not to disturb the pellet.
12.4 µl
?. [Illumina sequencing library preparation]
Prepare fragmentation/end prep mix, which will provide sufficient volume for one 96-well plate   AB1ReagentVolume (µl)2NEBNext Ultra II FS Reaction Buffer3363NEBNext Ultra II FS Reaction Enzyme964Total432Mix well by pipetting.
on ice
AB1ReagentVolume (µl)2NEBNext Ultra II FS Reaction Buffer3363NEBNext Ultra II FS Reaction Enzyme964Total432
?. [Illumina sequencing library preparation]
The Bravo will add  of fragmentation/end prep mix to each sample and mix by pipetting.
3.6 µl
?. [Illumina sequencing library preparation]
Seal and transfer the plate to a thermocycler with the heated lid set to  and run the following program:  AB1TemperatureTime272ºC15 minutes365ºC30 minutes44ºC∞
100 °C
AB1TemperatureTime272ºC15 minutes365ºC30 minutes44ºC∞
?. [Illumina sequencing library preparation]
Prepare adapter ligation mix, which will provide sufficient volume for one 96-well plate   AB1ReagentVolume (µl)2NEBNext Ultra II Ligation Master Mix14403NEBNext Ultra II Ligation Enhancer484TruSeq Duplexed Adapter (100 µM)125Nuclease-Free Water1086Total1608Mix well by pipetting.
on ice
AB1ReagentVolume (µl)2NEBNext Ultra II Ligation Master Mix14403NEBNext Ultra II Ligation Enhancer484TruSeq Duplexed Adapter (100 µM)125Nuclease-Free Water1086Total1608
?. [Illumina sequencing library preparation]
The Bravo will add  of ligation mix to each sample and mix by pipetting.
13.4 µl
?. [Illumina sequencing library preparation]
The plate is incubated on deck at  for  , however this step may also be performed on a thermocycler.
20 °C
?. [Illumina sequencing library preparation]
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.
?. [Illumina sequencing library preparation]
Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.
?. [Illumina sequencing library preparation]
Add 0.7 X volume of SPRI beads per sample ( SPRI :  amplified cDNA), mix well by pipetting.
20 µl
29.4 µl
?. [Illumina sequencing library preparation]
Incubate for  at
0 Room temperature
?. [Illumina sequencing library preparation]
Transfer the plate to the magnet, allow  for the beads to settle.
?. [Illumina sequencing library preparation]
Carefully remove and discard the supernatant without disturbing the bead pellet.
?. [Illumina sequencing library preparation]
Wash the beads with  80% freshly prepared ethanol for  remove ethanol and discard.
180 µl
?. [Illumina sequencing library preparation]
Repeat ethanol wash.
?. [Illumina sequencing library preparation]
Allow beads to dry
?. [Illumina sequencing library preparation]
Remove the plate from the magnet, add  nuclease-free water and resuspend by mixing well.
25 µl
?. [Illumina sequencing library preparation]
Incubate for at
0 Room temperature
?. [Illumina sequencing library preparation]
Transfer the plate to the magnet, allow  for the beads to settle.
?. [Illumina sequencing library preparation]
Transfer supernatant  into a new 96-well PCR plate, taking care not to disturb the pellet.
?. [Illumina sequencing library preparation]
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.
?. [Illumina sequencing library preparation]
Prepare PCR mix, which will provide sufficient volume for one 96-well plate    AB1ReagentVolume (µl)2KAPA HiFi HotStart ReadyMix30003Total3000
on ice
AB1ReagentVolume (µl)2KAPA HiFi HotStart ReadyMix30003Total3000
?. [Illumina sequencing library preparation]
The Bravo will add  PCR mix and  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.
25 µl
25 µl
?. [Illumina sequencing library preparation]
Seal and transfer the plate to a thermocycler with the heated lid set to  and run the following program:  ABC1TemperatureTimeCycles298ºC45 seconds1398ºC10 seconds8462ºC15 seconds572ºC3 minutes672ºC5 minutes174ºC∞1
100 °C
ABC1TemperatureTimeCycles298ºC45 seconds1398ºC10 seconds8462ºC15 seconds572ºC3 minutes672ºC5 minutes174ºC∞1
?. [Illumina sequencing library preparation]
PAUSE POINT amplified libraries can be stored at  for several weeks prior to library purification.
-20 °C
?. [Pooling and purification of amplified libraries]
In a post-PCR lab, use the Hamilton STAR or Beckman NX-8 to combine  of each sample per plate to form an equivolume pool of 96 samples.
5 µl
?. [Pooling and purification of amplified libraries]
Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.
?. [Pooling and purification of amplified libraries]
Manually transfer  of the equivolume pool into a 1.5 ml Eppendorf tube
400 µl
?. [Pooling and purification of amplified libraries]
Add 0.95 X volume of SPRI beads ( SPRI :  amplified libraries), mix well by pipetting.
380 µl
400 µl
?. [Pooling and purification of amplified libraries]
Incubate for  at
0 Room temperature
?. [Pooling and purification of amplified libraries]
Transfer the tube to a magnet, allow  for the beads to form a pellet.
?. [Pooling and purification of amplified libraries]
Carefully remove and discard the supernatant without disturbing the bead pellet.
?. [Pooling and purification of amplified libraries]
Wash the beads with  80% freshly prepared ethanol for  remove ethanol and discard.
180 µl
?. [Pooling and purification of amplified libraries]
Repeat ethanol wash.
?. [Pooling and purification of amplified libraries]
Allow beads to dry
?. [Pooling and purification of amplified libraries]
Remove the tube from the magnet, add  nuclease-free water and resuspend by mixing well.
400 µl
?. [Pooling and purification of amplified libraries]
Incubate for at
0 Room temperature
?. [Pooling and purification of amplified libraries]
Transfer tube to magnet, allow  for the beads to form a pellet.
?. [Pooling and purification of amplified libraries]
Transfer supernatant into a new tube, taking care not to disturb the pellet.
?. [Quality control and normilisation of sequencing libraries]
Library sequencing pools are quantified on an Agilent Bioanalyzer. Pools are then diluted to 2.8 nM for sequencing.
?. [Quality control and normilisation of sequencing libraries]
Run  of the library pool in triplicate on a Bioanalyzer using the DNA 1000 kit.
1 µl
?. [Quality control and normilisation of sequencing libraries]
Taking an average of the readings add nuclease-free water to the library pool to produce a final concentration of 2.8 nM.
?. [Sequencing]
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.