Feb 08, 2024

Public workspaceSingle Cell Sequencing 10x Chromium 5’ VDJ

DOI
dx.doi.org/10.17504/protocols.io.e6nvwdr77lmk/v1
  • Travis Dawson1,
  • Robert Marvin1,
  • Grace Chung1
  • 1Icahn School of Medicine at Mount Sinai
Sojin Kim
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwdr77lmk/v1
Protocol Citation: Travis Dawson, Robert Marvin, Grace Chung 2024. Single Cell Sequencing 10x Chromium 5’ VDJ. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdr77lmk/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: Working
We use this protocol and it's working
Created: February 08, 2024
Last Modified: February 08, 2024
Protocol Integer ID: 94914
Abstract
Single cell RNA sequencing technique (scRNA-Seq) obtains gene expression profiles of individual cells for analysis, as opposed to comparing averaged gene expression signals between bulk samples of cells. The ability to examine transcriptional changes between individual cells at a high resolution uniquely define rare cell populations, identify heterogeneity within cell populations, investigate cell population dynamics in depth over time, and even interrogate cell signaling pathways.

ScRNA sequencing enables the exploration of the cellular heterogeneity from the various biospecimen tissues; tumor, biopsy samples and organoid cells. To profile the immune repertoire of cells, full-length (5’ UTR to constant region), paired T-cell receptor (TCR) and/or B-cell receptor (BCR) transcripts from 500-10,000 individual cells per sample can be assessed. A pool of approximately 750,000 barcodes are sampled separately to index each cell’s transcriptome and antigen specificity. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10x Barcode. Libraries are generated and sequenced and 10x Barcodes are used to associate individual reads back to the individual partitions.
Materials
  • Chromium Next GEM Single Cell 5′ Library and Gel Bead Kit v1.1 16 rxns (10x Genomics #1000165)
  • Chromium Single Cell 5’ Library Construction Kit 16 rxns (10x Genomics #1000020)
  • Chromium Single Cell V(D)J Enrichment Kit, Human B Cell 96 rxns (10x Genomics #1000016)
  • Chromium Single Cell V(D)J Enrichment Kit, Human T Cell 96 rxns (10x Genomics #1000005)
  • Single Index Kit T Set A 96 rxns (10x Genomics #1000213)
  • Chromium Next GEM Chip G Single Cell Kit, 48 rxns (10x Genomics #1000202)
  • Chromium Controller & Next GEM Accessory Kit (#1000202)
  • Thermocycler - Veriti 96 well (Thermo Fisher Scientific #4375786)
  • Thermomixer 1.5 mL (Eppendorf #EP5382000023)
  • Quant Studio 3 (Thermo Fisher Scientific #A28567)
  • 2100 Bioanalyzer (Agilent #G2939BA)
  • 4200 Tape Station (Agilent #G2991A)
  • Nuclease Free Water (Ambion #AM9937)
  • Ethanol (Acros Organics #AC615095000)
  • Elution Buffer (Qiagen #19086)
  • Tween 20 (Thermo Fisher Scientific #BP337-100)
  • SPRISelect (Beckman Coulter #B23319)
  • 50% GLYCEROL (RICCA CHEMICAL, CAT# 329032)
  • DNA LOBIND TUBE 1.5 ML (EPPENDORF #022431021)
  • PCR TUBE STRIPS 0.2 ML (EPPENDORF #951010022)
Safety warnings
  • All personnel must have completed the necessary training, including annual refresher training, on the safe handling of potentially infectious material.
  • Personal protective equipment (PPE), which includes gowns, gloves, and protective goggles.
GEM Generation and RT Reaction
GEM Generation and RT Reaction
Prepare master mix on ice. Pipette mix 15x and centrifuge briefly.



Add 37.2 μl Master Mix into each tube of a PCR 8-tube strip on ice.
Assemble Chromium Next GEM Chip G:



Load 50% Glycerol into Unused Chip Wells (if <8 samples per chip): i. 70 ul to unused wells in row labeled 1. ii. 50 ul to unused wells in row labeled 2. iii. 45 ul to unused wells in row labeled 3.
Prepare Master Mix + Cell Suspension. Refer to the Cell Suspension Volume Calculator Table. Add the appropriate volume of nuclease-free water and corresponding volume of single cell suspension to Master Mix for a total of 75 ul in each tube. Gently pipette mix the cells suspension before adding to the Master Mix.
Gently pipette mix the Master Mix + Cell Suspension and using the same pipette tip, dispense 70 ul Master Mix + Cell Suspension into the bottom center of each well in row labeled 1 without introducing bubbles.
Snap the tube strip holder with the Gel Bead strip into a 10x Vortex Adapter. Vortex 30 sec. Centrifuge the Gel Bead strip for ~5 seconds. Confirm there are no bubbles at the bottom of the tubes and the liquid levels are even. Place the Gel Bead strip back in the holder. Secure the holder lid.
Puncture the foil seal of the Gel Bead tubes. Slowly aspirate 50 ul Gel Beads. Dispense into the wells in row labeled 2 without introducing bubbles. Wait 30 seconds.
Dispense 45 ul Partitioning Oil into the wells in row labeled 3 from a reagent reservoir. Failure to add Partitioning Oil to the top row labeled 3 will prevent GEM generation and can damage the Chromium Controller.
Attach 10x gasket (notch on top left)
Place the assembled chip with the gasket into the tray of the Chromium Controller, ensuring the chip stays horizontal. Close the tray and press the play button to begin run (18 minutes). Complete next steps during run.
Place a PCR 8-tube strip on ice.
When chip run is complete, press the eject button of the Controller to remove the chip. Discard the gasket, open chip holder, and fold the lid back until it clicks to expose the wells at a 45-degree angle.
Transfer 100 μl of GEMs into PCR strip tube. Pipette slowly. It should take ~20 seconds to pipette GEMs.
If multiple chips are run back-to-back, cover the GEM-containing strip tube and place on ice for no more than 1 hour.
Run the following thermocycler program:


NOTE: Store at 4°C for up to 72 h or at −20°C for up to a week, or proceed to the next step.
Post GEM Incubation Cleanup
Post GEM Incubation Cleanup
Add 125 ul Recovery Agent to each sample at room temperature. DO NOT pipette mix or vortex the biphasic mixture. Gently invert tube 10x to mix. Centrifuge briefly.
Slowly remove and discard 125 ul Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample.
Prepare Dynabeads Cleanup Mix:



Vortex and add 200 μl of Dynabeads Cleanup Mix to each sample. Pipette mix 5x (pipette set to 200 ul).
Incubate 10 min at room temperature.
Prepare Elution Solution I. Vortex and centrifuge briefly:



At the end of 10 min incubation, place on the 10x Magnetic Separator, high position (Magnet – High) until the solution clears.
Remove the supernatant.
Add 300 ul freshly prepared 80% ethanol to the pellet while on the magnet – High. Wait 30 seconds.
Remove the ethanol.
Add 200 ul 80% ethanol to the pellet. Wait 30 seconds.
Remove the ethanol.
Centrifuge briefly. Place on the magnet – Low.
Remove the remaining ethanol.
Remove from the magnet. Immediately add 35.5 ul Elution Solution I to avoid clumping.
Pipette mix 15x (pipette set to 30 ul) without introducing bubbles.
Incubate 2 minutes at room temperature
Centrifuge briefly. Place on the magnet – Low until the solution clears.
Transfer 35 ul sample to a new tube strip.
cDNA Amplification
cDNA Amplification
Prepare cDNA Amplification Mix on ice. Vortex and centrifuge briefly.



Add 65 ul cDNA Amplification Reaction Mix to 35 ul sample. Pipette mix 15x (pipette set to 90 ul). Centrifuge briefly.
Incubate in a thermal cycler with the following protocol. NOTE: Samples can be stored at 4°C up to 72 hours or at -20°C for up to 1 week or proceed to the next step.



Ensure sample volume is 100 μl (bring up to 100 μl with Buffer EB if needed). Vortex to resuspend the SPRIselect reagent. Add 60 ul SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 150 ul).
Incubate 5 min at room temperature.
Place on the magnet – High until the solution clears (~5 minutes).
Remove the supernatant.
Add 200 ul of 80% ethanol to the pellet. Wait 30 sec.
Remove the ethanol.
Repeat steps 43 and 44 for a total of 2 washes.
Centrifuge briefly and place on the magnet – High.
Remove any remaining ethanol. Air dry for 2 minutes. DO NOT exceed 2 minutes as this will decrease elution efficiency.
Remove from magnet. Add 45.5 ul Buffer EB. Pipette mix 15x.
Incubate 2 min at room temperature.
Place the tube strip on the magnet – High until the solution clears (~2 minutes).
Transfer 45 ul sample to a new tube strip and *Label the top of tubes with "cDNA". NOTE: Store at 4°C for up to 72 hours or at −20°C for up to 4 weeks or proceed to the next step.
cDNA QC & Quantification
cDNA QC & Quantification
Run 1 ul of sample on Qubit dsDNA HS Assay.
Either run 1 ul of 1 ng/ul diluted sample on an Agilent Bioanalyzer High Sensitivity chip to determine fragment size.
OR run 2 ul of 1 ng/ul diluted sample on the Agilent Tapestation High Sensitivity D1000 ScreenTape to determine fragment size.



Gene Expression Library Construction
Gene Expression Library Construction
Initiate the following thermocycler protocol to pre-cool the block.


Prepare Fragmentation Mix on ice. Pipette mix and centrifuge briefly.





Determine the volume for 50 ng mass of sample. Dispense the sample volume in a tube strip on ice. If the volume required for 50 ng is less than 20 μl, adjust the total volume of each sample to 20 μl with nuclease-free water. If the volume for 50 ng exceeds 20 μl, carry ONLY 20 μl sample into library construction.
Aliquot 30 μl of Fragmentation Mix to each well, mix 15x and spin down before returning to ice, let cool for 1 minute.
Transfer to pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the protocol.
After the cycle is complete, vortex to resuspend SPRIselect reagent. Add 30 ul SPRIselect (0.6X) reagent to each sample. Pipette mix 15x (pipette set to 75 ul).
Incubate 5 min at room temperature.
Place on the magnet – High until the solution clears (~5 minutes). DO NOT discard the supernatant.
Transfer 75 ul supernatant to a new tube strip.
Vortex to resuspend SPRIselect reagent. Add 10 ul SPRIselect reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 80 ul).
Incubate 5 min at room temperature.
Place on the magnet – High until the solution clears.
Remove 80 ul supernatant, making sure not to discard any beads.
Add 125 ul 80% ethanol to the pellet. Wait 30 seconds.
Remove the ethanol.
Repeat steps 68 and 69 for a total of 2 washes.
Centrifuge briefly and place on the magnet – Low until the solution clears. Remove remaining ethanol. DO NOT over dry to ensure maximum elution efficiency.
Remove from the magnet. Add 50.5 ul Buffer EB to each sample. Pipette mix 15x.
Incubate 2 min at room temperature.
Place on the magnet – High until the solution clears.
Transfer 50 ul sample to a new tube strip.
Prepare Adaptor Ligation Mix on ice. Pipette mix and centrifuge briefly.



Add 50 μl of Adaptor Ligation Mix to 50 ul sample. Pipette mix 15x (pipette set to 90 ul). Centrifuge briefly.
Incubate in a thermal cycler with the following protocol.



Vortex to resuspend SPRIselect Reagent. Add 80 ul SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 150 ul).
Incubate 5 min at room temperature.
Place on the magnet – High until the solution clears.
Remove the supernatant.
Add 200 ul 80% ethanol to the pellet. Wait 30 sec.
Remove the ethanol.
Repeat steps 83 and 84 for a total of 2 washes.
Centrifuge briefly. Place on the magnet – Low.
Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
Remove from the magnet. Add 30.5 ul Buffer EB. Pipette mix 15x.
Incubate 2 min at room temperature.
Place on the magnet – Low until the solution clears.
Transfer 30 ul sample to a new tube strip.
Choose the appropriate sample index set to ensure that no sample indices overlap in a multiplexed sequencing run. Record the 10x sample index name (PN-2000240 Single Index Plate T Set A well ID) used.
Prepare Sample Index PCR Mix:



Add 60 ul Sample Index PCR Mix to 30 ul sample.
Add 10 ul of an individual Single Index to each well and record the well ID used. Pipette mix 5x (pipette set to 90 ul). Centrifuge briefly.
Incubate in a thermal cycler with the following protocol. NOTE: Store at 4°C for up to 72 hours or at −20°C for up to 4 weeks or proceed to the next step.



Vortex to resuspend the SPRIselect reagent. Add 60 ul SPRIselect Reagent (0.6X) to each sample. Pipette mix 15x (pipette set to 150 ul).
Incubate 5 minutes at room temperature.
Place on the magnet – High until the solution clears (~2 minutes).
Discard the supernatant
Add 200 ul 80% ethanol to the pellet. Wait 30 seconds.
Remove the ethanol.
Repeat steps 101 and 102 for a total of 2 washes.
Centrifuge briefly. Place on the magnet – Low.
Remove remaining ethanol.
Remove from the magnet. Immediately add 35.5 ul Buffer EB. Pipette mix 15x
Incubate 2 minutes at room temperature.
Centrifuge briefly. Place on the magnet – Low.
Transfer 35 ul sample to a new tube strip. NOTE: Samples can be stored at 4°C up to 72 hours or -20°C for long-term storage.
V(D)J Target Enrichment from cDNA
V(D)J Target Enrichment from cDNA
Add 33ul of Nuclease-Free water to PCR tube on ice.
Add 2ul of cDNA Amp product to water.
Prepare Target Enrichment 1 master mix on ice.



Add 65ul of master mix to each sample, mix 5x on ice.
Run the following thermocycler conditions. NOTE: Samples can be stored at 4°C up to 72 hours before the next step.


Vortex to resuspend SPRIselect Reagent. Add 80 ul SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 150 ul).
Incubate 5 min at room temperature.
Place on the magnet – High until the solution clears.
Remove the supernatant.
Add 200 ul 80% ethanol to the pellet. Wait 30 sec.
Remove the ethanol.
Repeat steps 10 and 11 for a total of 2 washes.
Centrifuge briefly. Place on the magnet – Low.
Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
Remove from the magnet. Add 30.5 ul Buffer EB. Pipette mix 15x.
Incubate 2 min at room temperature.
Place on the magnet – Low until the solution clears.
Transfer 30 ul sample to a new tube strip. NOTE: Samples can be stored at 4°C up to 72 hours or -20°C up to 1 week before the next step.
Prepare Target Enrichment 2 master mix on ice.



Add 65ul of master mix to each sample, mix 5x on ice.
Run the following thermocycler conditions. NOTE: Samples can be stored at 4°C up to 72 hours before the next step.


Vortex to resuspend SPRIselect Reagent. Add 50 ul SPRIselect Reagent (0.8X) to each sample and pipette mix 15x (pipette set to 145 ul).
Incubate 5 min at room temperature.
Place on the magnet – High until the solution clears. DO NOT discard the supernatant.
Transfer 145 μl supernatant to a new tube strip.
Add 30 μl of SPRIselect Reagent, mix 15x, incubate 5 min.
Place on the magnet – High for 5 min., then remove 170 μl of supernatant.
Add 200 ul 80% ethanol to the pellet. Wait 30 sec.
Remove the ethanol.
Repeat steps 28 and 29 for a total of 2 washes.
Centrifuge briefly. Place on the magnet – Low.
Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
Remove from the magnet. Add 45.5 ul Buffer EB. Pipette mix 15x.
Incubate 2 min at room temperature.
Place on the magnet – Low until the solution clears.
Transfer 45 ul sample to a new tube strip. NOTE: Samples can be stored at 4°C up to 72 hrs. or -20°C up to 1 week before the next step.
V(D)J Target Enrichment Library Construction
V(D)J Target Enrichment Library Construction
Initiate following thermocycler protocol to pre-cool block:


Prepare Fragmentation Mix on ice


Determine the volume for 50 ng mass of sample (see example calculation at step 3.3). Dispense the sample volume in a tube strip on ice. If the volume required for 50 ng is less than 20 μl, adjust the total volume of each sample to 20 μl with nuclease-free water. If the volume for 50 ng exceeds 20 μl, carry ONLY 20 μl sample into library construction.
Aliquot 30 μl of Fragmentation Mix to each well, mix 15x and spin down before returning to ice, let cool for 1 minute.
Transfer to pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the protocol.
Prepare Adaptor Ligation Mix on ice.


Remove tube strip containing Fragmentation Rxn from the thermocycler.
Add 50 μl of Adaptor Ligation Mix to each sample, mix 15x, and spin briefly.
Run the following thermocycler program:


Remove tube from thermocycler. Add 80 μl of SPRIselect Reagent, mix 15x, incubate 5 minutes, complete next step during incubation.
Prepare Sample Index PCR Mix on ice.


Place on the magnet – High until the solution clears.
Remove the supernatant...
Add 200 ul 80% ethanol to the pellet. Wait 30 sec.
Remove the ethanol.
Repeat steps 159 and 160 for a total of 2 washes.
Centrifuge briefly. Place on the magnet – Low.
Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
Remove from the magnet. Add 30.5 ul Buffer EB. Pipette mix 15x.
Incubate 2 min at room temperature.
Place on the magnet – Low until the solution clears.
Transfer 30 ul sample to a new tube strip.
Add 60 μl of Sample Index PCR Mix to each sample
Add 10 μl of an individual sample index (Single Index Plate T Set A) to each well and record their assignment.
Mix by pipetting 15x and spin briefly. Run the following thermocycler program:NOTE: Samples can be stored at 4°C up to 72 hours before the next step


Vortex to resuspend SPRIselect reagent. Add 80 ul SPRIselect reagent to each sample. Pipette to mix 15x.
Incubate 5 minutes at room temperature.
Place on the magnet – High until the solution clears (~2 minutes).
Discard the supernatant
Add 200 ul 80% ethanol to the pellet. Wait 30 seconds.
Remove the ethanol.
Repeat steps 175 and 176 for a total of 2 washes.
Centrifuge briefly. Place on the magnet – Low.
Remove remaining ethanol.
Remove from the magnet. Immediately add 35.5 ul Buffer EB. Pipette mix 15x
Incubate 2 minutes at room temperature.
Centrifuge briefly. Place on the magnet – Low.
Transfer 35 ul sample to a new tube strip. NOTE: Samples can be stored at 4°C up to 72 hours or -20°C for long-term storage.
LIBRARY QUANTIFICATION
LIBRARY QUANTIFICATION
Qubit: Run 1 ul sample at 1:5 dilution on the Qubit dsDNA HS Assay Kit
Bioanalyzer/Tapestation:
  1. EITHER Run 1 ul sample diluted to 3 ng/ul on the Agilent Bioanalyzer High Sensitivity DNA chip to determine fragment size. Lower molecular weight product (<150 bp) may be present. This does not affect sequencing.
  2. OR Run 2 ul sample diluted to 1 ng/ul on the Agilent Tapestation High Sensitivity D1000 ScreenTape to determine fragment size.


qPCR


Thaw KAPA Library Quantification Kit for Illumina Platforms
Dilute 1 ul sample with deionized water to appropriate dilutions that fall within the linear detection range of the KAPA Library Quantification Kit for Illumina Platforms. (For more accurate quantification, make the dilution(s) in duplicate).
Make enough Quantification Master Mix for the DNA dilutions per sample and the DNA Standards (plus 10% excess) using the guidance for 1 reaction volume below:


Dispense 16 ul Quantification Master Mix for sample dilutions and DNA Standards into a 96 well PCR plate.
Incubate in a thermal cycler with the following protocol.


Follow the manufacturer’s recommendations for qPCR-based quantification. For library quantification for sequencer clustering, determine the concentration using the average size in the region of 175 – 1,000 bp for gene expression libraries.
SEQUENCING
SEQUENCING
Sequencing Libraries: Chromium Single Cell V(D)J Enriched libraries, 5' Gene Expression libraries, and Cell Surface Protein libraries comprise standard Illumina paired-end constructs which begin with P5 and end with P7. These libraries include 16 bp 10x Barcodes encoded at the start of TruSeq Read 1. Sample index sequences are incorporated as the i7 index read for V(D)J Enriched and 5' Gene Expression libraries; as i7 index read N for Cell Surface Protein library. TruSeq Read 1, TruSeq Read 2, and Nextera Read 2 (Read 2N) are all standard Illumina sequencing primer sites. TruSeq Read 1 and TruSeq Read 2 are used in paired-end sequencing of V(D)J Enriched and 5' Gene Expression libraries. TruSeq Read 1 and Nextera Read 2 (Read 2N) are used for paired-end sequencing of Cell Surface Protein library. Sequencing these libraries produce a standard Illumina BCL data output folder.


Illumina Sequencer Compatibility
The compatibility of the listed sequencers has been verified by 10x Genomics. Some variation in assay performance is expected based on sequencer choice.
MiSeq
NextSeq 500/550 (High Output)
HiSeq 2500 (Rapid Run)
HiSeq 3000.4000
NovaSeq
Sample Indices
Each sample index in the Chromium i7 Sample Index Kit (PN-120262) and Chromium i7 Sample Index Kit Plate N, Set A (PN-1000084) is a mix of 4 different sequences to balance across all 4 nucleotides. If multiple samples are pooled in a sequence lane, the sample index name (i.e. the Chromium i7 Sample Index plate well ID) is needed in the sample sheet used for generating FASTQs with “cellranger mkfastq”. If multiple libraries are pooled in a sequence lane, a separate sample index is needed with each library.
Sequencing Depth & Run Parameters


Library Pooling
The 3ʹ Gene Expression libraries maybe pooled for sequencing, taking into account the differences in cell number and per-cell read depth requirements between each library. Samples utilizing the same sample index should not be pooled together, or run on the same flow cell lane, as this would not enable correct sample demultiplexing.


Protocol references
None.