TITLE: Massively parallel long-read sequencing of single cell RNA isoforms
AUTHORS: Jafar S Jabbari, Luyi Tian

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the subsampling of 10x Chromium generated single cell GEMs after reverse transcription for cDNA amplification. The protocol enables transcriptome sequencing of full-length cDNA from a flexible  number of single cells captured on Chromium using long-read sequencers such as Nanopore and PacBio, in addition to preparing short-read Illumina libraries. The protocol is compatible with 10x Single Cell 3' and 5' Gene Expression kits including feature barcoding and also other 10x compatible multiomics methodologies such as CITE-seq.</div></div>

[STEPS]
?. [GEM Generation & Barcoding]
?. [GEM Generation & Barcoding]
Follow  the 10x Genomics user guide for Single Cell Gene Expression (v3) or Single Cell Immune Profiling (v1 Chemistry) kits for GEM Generation and Barcoding for the required number of cells.NOTE: This protocol is also compatible with Next Gem single cell 3’ (v3.1 Chemistry) and Single Cell Immune Profiling (v1.1 Chemistry) and also feature barcoding.
?. [GEM Generation & Barcoding]
After transferring GEMs perform GEM-RT Incubation according to the user guide.NOTE: In some experiments we have extended RT time up to 2 hours to potentially increase the number of longer transcripts and have not observed any adverse effect. However, we have not systematically investigated if increased incubation time results in higher number of longer transcripts.
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
Prepare Sub-Sampling Cleanup Mix:Add 2.5 µl Additive A to 97.5 µl EB (v1 chemistry)Or Add 2 µl Reducing Agent B to 98 µl EB (v3 chemistry).
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
Transfer 125 µl Recovery Regent into a fresh well of a 8-strip tube.
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
Dispense 63 µl of Sub-Sampling Cleanup Mix into well containing Recovery Reagent.
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
Transfer 10-20 µl of GEMs from the middle of the opaque emulsion layer into the Recovery Agent containing the Sub-Sampling Cleanup Mix.NOTE: This proportion can be adjusted to sub-sample required number of cells. For GEM volumes above 20 μl reduce Sub-Sampling Cleanup Mix volume by 0.7 μl for every 1 μl at step 2.3 and add that volume to remaining GEMs at step 2.7.
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
Using the same pipette, pipette-mix several times to break the emulsions.
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
Slowly remove 125 µl of Recovery Agent (pink) from the bottom according to the 10x user guide.
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
The Post GEM-RT Cleanup step with Dynabeads MyOne SILANE is done according to the v1 or v3 user guide for both fractions.NOTE: Each fraction will be treated as a separate sample and will follow the standard 10x protocol for the remaining steps. In addition to standard protocol, the sub-sample will go through long-read template preparation (Step 5).
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
Set up cDNA Amplification reactions for both fractions according to the user guide.NOTE: Identical reagents from Chromium Single Cell 5’ Library Construction Kit can be used for extra cDNA amplification (Kit dependent).
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
Adjust the PCR cycle numbers for each fraction based on cell number and carry out cleanup as per the 10x user guide.NOTE: In some experiments we have increased extension time to 3 min to allow efficient extension of longer cDNA fragments but we have not quantified the effect comparatively.
?. [Gene Expression Library Construction]
?. [Gene Expression Library Construction]
Carry out Gene Expression Library Construction for the main sample and the sub-sample separately according to the user guide.NOTE: Identical reagents from Chromium Single Cell 5’ Library Construction Kit can be used for extra library prep.
?. [Sequencing short-read Illumina Libraries]
?. [Sequencing short-read Illumina Libraries]
Sequencing is performed according to the 10x user guide recommendations.
?. [Template Preparation for Long-Read Sequencing]
?. [Template Preparation for Long-Read Sequencing]
Optimise cDNA PCR for long-read library preparation.  AB1  Reagent    Amount  25x PrimeSTAR GXL Buffer (Mg2+ plus)  10 µl  3dNTP Mixture (2.5 mM each)  4 µl  4FPSfiIA (10 µM)1 µl5RPSfiIBr (10 µM)1 µl6Subsample cDNA0.5-1 ng7Nuclease-free Water33 µl - volume of subsample cDNA8PrimeSTAR GXL DNA Polymerase  1 µl  9  Total    50 µl   In a thermocycler with heated lead, incubate as follows:  ABCD1  Step  Temperature (°C)  Time     Cycles  2Initial Denaturation98  30 sec    1  3Denaturation9810 sec8-14 (see below)4Annealing6515 sec5Extension688 min6Final extension685 min
AB1  Reagent    Amount  25x PrimeSTAR GXL Buffer (Mg2+ plus)  10 µl  3dNTP Mixture (2.5 mM each)  4 µl  4FPSfiIA (10 µM)1 µl5RPSfiIBr (10 µM)1 µl6Subsample cDNA0.5-1 ng7Nuclease-free Water33 µl - volume of subsample cDNA8PrimeSTAR GXL DNA Polymerase  1 µl  9  Total    50 µl
ABCD1  Step  Temperature (°C)  Time     Cycles  2Initial Denaturation98  30 sec    1  3Denaturation9810 sec8-14 (see below)4Annealing6515 sec5Extension688 min6Final extension685 min
?. [Template Preparation for Long-Read Sequencing]
After 8 cycles take 5 µl out for analysis.
?. [Template Preparation for Long-Read Sequencing]
Repeat from Denaturation for 2 extra cycles and take 5 µl out and continue extra 2 cycles up to 14.
?. [Template Preparation for Long-Read Sequencing]
Quantify 2 µl of each sample taken from different cycles with Qubit dsDNA High Sensitivity assay.
?. [Template Preparation for Long-Read Sequencing]
Dilute each sample to 1 ng/µl and run on HSD5000 Tape.
?. [Template Preparation for Long-Read Sequencing]
The optimum number of cycles is that which yields at least 6 ng/µl cDNA without changing the peak distribution in the input cDNA. The following shows a comparison of input cDNA to PCR runs for 10-14 cycles on HSD5000 Tape. In this case all cycles met the peak distribution criteria and we chose 12 cycles for cDNA bulking.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Set up 5 PCR reactions and cycle them to the optimum number obtained from optimisation step.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Pool PCR reactions and add 0.6x SpriSelect beads and mix.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Incubate at room temperature for 5 min.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Place on the magnet until the solution clears.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Incubate for 2 min at room temperature.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Place the tube on the magnet until the solution clears.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Quantify 1 µl with Qubit dsDNA HS assay.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
QC amplified cDNA by running a 1 ng/µl dilution on HSD5000 Tape.
?. [Sequencing Full-Length cDNA]
?. [Sequencing Full-Length cDNA]
Prepare library for Nanopore or PacBio sequencing according to the relevant protocol.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Remove the supernatant.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
To wash add 200 µl freshly prepared 80% ethanol to the pellet. Wait 30 sec.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Remove the ethanol.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Repeat wash step for a total of 2 washes.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Centrifuge briefly and place on the magnet then remove any remaining ethanol.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Air dry for 2 min. Avoid overdrying (beads become cracked).
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Remove from the magnet. Add 50 µl Buffer EB. Pipette mix 15x.
?. [Generating a Sufficient Amount of cDNA for Long-Read Library Preparation]
Transfer 49 µl of solution to a new tube, without disturbing the beads.
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
Process remaining GEMs (still in emultion layer) as per the 10x user guide.
?. [Sub-Sampling, Post GEM-RT Cleanup and cDNA Amplification]
After cleanup save 5-10 ng of the sub-sampled  cDNA for long-read template preparation according to step 5 of this protocol. The remainder of the sub-sampled cDNA and main cDNA sample are utilised for short-read sequencing in steps 3 and 4 of this protocol.NOTE: Fractions for feature barcoding (if used) will be sequenced by short-read sequencing only.
?. [Sequencing Full-Length cDNA]
Sequencing depth depends on the number of cells captured, sample source, required resolution and sequencing platform. UMI counts from analysis of Illumina reads can be used as a guide. With current chemistry 25% of Nanopore and 65% of PacBio reads can be assigned to cells.