May 26, 2022

Public workspace10X Genomics Single-Nucleus Multiome (RNA + ATAC) Assay for Profiling Adult Human Tissues V.2

DOI
dx.doi.org/10.17504/protocols.io.5qpvoby69l4o/v2
  • 1University of California, San Diego;
  • 2Washington University School of Medicine, Saint Louis
Blue Lake
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DOI: dx.doi.org/10.17504/protocols.io.5qpvoby69l4o/v2
External link: http://genome-tech.ucsd.edu/ZhangLab/
Protocol Citation: Kimberly Conklin, Bo Zhang, Amanda Knoten, Dinh Diep, Blue Lake, Sanjay Jain, Kun Zhang 2022. 10X Genomics Single-Nucleus Multiome (RNA + ATAC) Assay for Profiling Adult Human Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoby69l4o/v2Version created by Blue Lake
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: May 26, 2022
Last Modified: May 26, 2022
Protocol Integer ID: 63303
Keywords: Kidney, KPMP, Single Nucleus, RNA Sequencing, 10X Genomics, Chromatin Accessibility, ATAC
Abstract
10X Genomics Single Cell 3' (v3) RNA sequencing is a microdroplet-based method that permits the effective capture and sequencing of the mRNA and pre-mRNA molecules from single nuclei [1]. RNA molecules are transcribed and processed within the nucleus before exporting to ER for translation into proteins. As such, nuclear RNA is a mixture of nascent transcripts, partially or fully processed mRNA, and various non-coding RNA molecules. The total RNA content within the nucleus is roughly 10% of the RNA content in a whole cell, but has been found to accurately represent whole cell expression values in adult human tissues [2,3] including the kidney [4]. 10x Genomics Single Cell ATAC sequencing is a microdroplet-based method that allows for the effective capture, sequencing and profiling of accessible chromatin in single nuclei. Chromatin accessibility is a major determinant of gene regulation, defining the transcriptional regulatory networks that determine cellular identity and function as well as additional biological processes (e.g., differentiation, proliferation, development and responses to the extracellular environment). The 10X Multiome ATAC + Gene Expression assay permits capture of both RNA expression and epigenomic profiles from the same nuclei for a deeper understanding of cell type or state gene regulation. Here we present a modified version of the published 10X protocol [5] that we have adapted for the processing of nuclei isolated from adult human organs or tissues (e.g. kidney).

Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can effectively circumvent the non-uniform or incomplete dissociation of solid tissues into single cells, as well as RNA degradation or artefacts (such as stress response) during dissociation. This 10X Multiome ATAC + Gene Expression protocol permits the generation of a molecular atlas of a human organ or tissue with comprehensive cell types and minimal processing artifacts.


References
1. Chromium Single Cell 3' Reagent Kits v3 User Guide (Rev A) CG000183, support.10xgenomics.com.
2. Lake et al. (2016). Science, doi:10.1126/science.aaf1204.
3. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038.
4. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2.
5. Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338 Rev D).
Guidelines
Full protocol is from 10X Genomics. All modifications are to the original protocol (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (Rev D) CG000338, support.10xgenomics.com)
Materials
MATERIALS
ReagentChromium Next GEM Single Cell Multiome ATAC Gene Expression10x GenomicsCatalog #PN-1000283
ReagentChromium Next GEM Chip J Single Cell Kit10x GenomicsCatalog #PN-1000234
ReagentSingle Index Kit N Set A10x GenomicsCatalog #1000212
ReagentDual Index Kit TT Set A10x GenomicsCatalog #1000215
Nuclease-free water
Ethanol (200 proof)
SPRIselect reagent set
50% glycerol
10% Tween 20
Low TE Buffer (10mM Tris-HCl, pH 8.0, 0.1 EDTA)
Qiagen buffer EB
Qubit dsDNA HS Assay Kit
PCR strip tubes with cap
LoBind 1.5 ml tubes
Qubit Assay tubes
Isolate Nuclei
Isolate Nuclei
Prepare nuclei according to the protocol "Isolation of single nuclei from solid tissues" steps 1-14, with the following modifications:
  • Tissue sections are cut and stored on dry ice until processed for nuclei isolation (locally or shipped/stored overnight). Sections cannot be stored in RNAlater or in a -80C freezer.
  • Step 1: replace Enzymatics RNAse Inhibitor with Protector RNase Inhibitor (Sigma-Aldrich, Catalog #3335402001), increase concentration of RNAse Inhibitor from 0.04 U/µl to 1.0 U/µl, add cOmplete protease inhibitor cocktail (Roche, cat #11836153001) for final 1X concentration and exclude DAPI.
  • Step 4: To PBSE buffer, add 1 tablet of cOmplete protease inhibitor cocktail to 50 µl PBSE.
  • Step 13: Determine total amount of wash buffer needed and add 0.1% Protector RNase Inhibitor (Sigma, PN-3335399001) to volume of PBSE + cOmplete protease inhibitor.
Resuspend nuclei in Amount30 µL to Amount100 µL of 1X Diluted Nuclei Buffer (20X nuclei buffer, PN2000207, provided by 10x Genomics), volume depends on target concentration. 1X Diluted Nuclei Buffer is prepared following the user guide Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (CG000338 Rev D).

Count nuclei (e.g. BioRad T20 Cell Counter) .
Check nuclei integrity under fluorescent microscope using DAPI channel. Nuclei should appear distinct, have rounded borders and the majority occurring as singlets. High clumping rates would indicate damaged nuclei and would require re-filtering using 30-µm CellTrics filter or exclusion.
Dilute nuclei stock to be in the 1,000-3,200 nuclei per µL range in Amount5 µL total volume using the dilution guide
Note
QC cutoff: minimum of 5,000 nuclei



Transposition
Transposition
Prepare Reagents for use
  1. Thaw, vortex, and centrifuge ATAC Buffer B.
  2. Centrifuge ATAC Enzyme B (maintain TemperatureOn ice ) before adding to Transposition Mix.

Prepare Transposition Mix.
  1. Prepare TemperatureOn ice . Pipette mix 10X and centrifuge briefly.
  2. Add Amount10 µL Transposition Mix to each tube of a PCR 8-tube strip TemperatureOn ice .
  3. Calculate the volume of Nuclei Stock and Diluted Nuclei Buffer for a total volume of Amount5 µL using the Nuclei Concentration Guidelines.
  4. Add the calculated volume of Diluted Nuclei Buffer to the Transposition Mix, pipette mix, and centrifuge briefly.
  5. Gently pipette mix the Nuclei Stock before adding the calculated volume of the Nuclei Stock to the tube containing the Transposition Mix. Gently pipette mix 6X (pipette set to 10 µl). DO NOT centrifuge.


Isothermal Incubation
  1. Incubate at Temperature37 °C for Duration01:00:00 in a thermal cycler with lid set at Temperature50 °C for transposition and addition of adapter sequences, which are added to the ends of the DNA fragments.
  2. Proceed immediately to next step.



1h
GEM Generation and Barcoding
GEM Generation and Barcoding
Prepare Reagents for use
  1. Equilibrate gel beads to TemperatureRoom temperature for Duration00:30:00 .
  2. Thaw, vortex, and centrifuge Barcoding Reagent Mix, Template Switch Oligo, and Reducing Agent B. Verify no precipitate in Reducing Agent B.
  3. Centrifuge Barcoding Enzyme Mix (maintain TemperatureOn ice ) before adding to Master Mix.


Prepare Master Mix
  1. PrepareTemperatureOn ice . Pipette mix 10X and centrifuge briefly.
  2. Keep TemperatureOn ice .
  3. Add Amount60.0 µL Master Mix to each tube containing Transposed Nuclei TemperatureOn ice .



Load Chromium Single Cell J Chip
1. Assemble Chromium Chip J in a 10X Chip Holder.
2. Must load chip in order according to row label for microfluidic channels to work properly: Row 1 → Row 2 → Row 3
3. Make sure NO bubbles are introduced while loading chip.
4. Dispense 50% glycerol solution into unused Chip Wells (if <8 samples used per chip)
a. Amount70 µL into unused wells in Row 1
b. Amount50 µL into unused wells in Row 2
c. Amount45 µL into unused wells in Row 3
5. Add Amount60.0 µL Master Mix to each tube containing Transposed Nuclei on ice. Gently pipette mix 5X.
6. Load Amount70 µL Master Mix and Transposed Nuclei into the bottom center of each well in Row 1 without introducing bubbles.
7. Snap the Gel Bead strip into a 10x Vortex Adapter. Vortex Duration00:00:30 . Remove the Gel Bead strip and centrifuge for ~ Duration00:00:05 . Confirm there are no bubbles at the bottom of the tubes and liquid levels look even.
8. Load Amount50 µL Gel Beads in Row 2. Dispense slowly and without introducing bubbles. Wait Duration00:00:30 .
9. Load Amount40 µL Partitioning Oil into wells of Row 3. Proceed immediately to the next step (and to running Chip in Chromium Controller).
10. Attach 10X gasket. Align the top-notch. Ensure gasket holes are aligned with the wells. Avoid touching the smooth gasket surface. Do not press down on the gasket.
1m 5s
Run the Chromium Controller
  1. Press the eject button on the Controller to eject the tray.
  2. Place the assembled chip with the gasket in the tray. Press the button to retract the tray.
  3. Confirm the program on screen. Press the play button.
  4. At completion of the run (~Duration00:18:00 ), the Controller will chime. Proceed immediately to the next step.

Safety information
Firmware Version 4.00 or higher is required in the Chromium Controller for use of this protocol.


18m
Transfer GEMs
  1. Chill a PCR 8-tube strip TemperatureOn ice .
  2. Press the eject button to remove the Chip.
  3. Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees.
  4. Check the volume in rows 1-3. Abnormally high volume in any well indicates a clog.
  5. Slowly aspirate 100 µL GEMs from the lowest points of the Recovery Wells without creating a seal between the pipette tips and the wells.
Note
GEMs should appear opaque and uniform across all channels. Excess partitioning oil (clear) in the pipette tips indicates a potential clog.

6. Slowly dispense (~Duration00:00:20 ) GEMs into the tube strip TemperatureOn ice with the pipette tips against the sidewalls of the wells.


GEM Incubation
  1. Incubate in a thermocycler for Duration01:15:00 to complete reverse transcription.
  2. During GEM incubation, equilibrate Quenching Agent to TemperatureRoom temperature .
  3. Upon completion of GEM incubation, proceed immediately to next step.
1h 15m
Quenching Reaction
  1. Add 5 µl Quenching Agent to each sample to stop the reaction.
  2. Slowly pipette mix 10X (pipette set to 90 µl). The solution will be viscous, ensure that no liquid remains along the the sidewalls of the tube or the pipette tips.
  3. Store at Temperature-80 °C for up to 4 weeks or proceed to the next step.
Post GEM Incubation Cleanup -- prepare reagents for use
  1. Thaw, vortex, and centrifuge a tube of Reducing Agent B.
  2. Thaw Cleanup Buffer at Temperature65 °C for Duration00:10:00 with shaking at max rpm on a thermomixer, then cool to room temperature before use. Verify there are no visible crystals.
Post GEM Incubation Cleanup -- Dynabeads
  1. Add Amount125 µL Recovery Agent to each sample at room temperature. DO NOT pipette mix or vortex. Gently invert tube 10X to mix and centrifuge briefly.
  2. Carefully aspirate Amount125 µL of pink oil phase from the bottom of the tube. DO NOT aspirate any aqueous sample.
  3. Prepare Dynabeads Cleanup Mix (follow recipe in User Guide).
  4. Vortex and add Amount200 µL Dynabeads Cleanup Mix to each sample. Pipette 10X to mix (pipette set to 200 µl).
  5. Incubate at room temperature for Duration00:10:00 with caps open.
  6. Prepare Elution Solution I (follow recipe in User Guide). Vortex and centrifuge briefly.
  7. After the 10-minute incubation period, place the samples on a magnetic separator. Wait until solution clears before removing the supernatant.
  8. Add Amount300 µL of freshly prepared 80% ethanol. Wait Duration00:00:30 before removing ethanol.
  9. Add Amount200 µL of freshly prepared 80% ethanol. Wait Duration00:00:30 before removing ethanol.
  10. Remove from magnet and immediately add Amount50 µL Elution Solution 1 to avoid clumping. Pipette mix (pipette set to 50 µl) without introducing bubbles.
  11. Incubate Duration00:01:00 at TemperatureRoom temperature .
  12. Centrifuge briefly and move back to magnet.
  13. After solution clears, transfer Amount50 µL to a new tube strip.
12m
Post GEM Incubation Cleanup -- SPRIselect
  1. Add Amount90 µL SPRIselect reagent to each sample and pipette mix thoroughly.
  2. Incubate Duration00:05:00 at TemperatureRoom temperature .
  3. Centrifuge briefly and place on magnet until solution clears. Remove supernatant.
  4. Add Amount200 µL freshly prepared 80% ethanol to pellet and wait Duration00:00:30 before removing ethanol.
  5. Repeat step 4 for a total of 2 washes.
  6. Centrifuge briefly and place on magnet.
  7. Remove any remaining ethanol (note: residual ethanol can inhibit Pre-Amplification PCR and impact assay performance).
  8. Remove from magnet and immediately add Amount46.5 µL Buffer EB. Pipette mix (pipette set to 45 µl) without introducing bubbles.
  9. Incubate Duration00:02:00 at TemperatureRoom temperature .
  10. Centrifuge briefly and return to magnet.
  11. After solution clears, transfer Amount46 µL to a new tube strip.
7m 30s
Pre-Amplification PCR & Cleanup
Pre-Amplification PCR & Cleanup
Pre-Amplification PCR
  1. Thaw, vortex, and centrifuge Pre-Amp Primers.
  2. Centrifuge Pre-Amp Mix (maintain TemperatureOn ice ) before adding.
  3. Prepare Pre-Amplification Mix TemperatureOn ice (follow recipe in User Guide). Pipette mix 10X and centrifuge briefly.
  4. Add Amount54 µL Pre-Amplification Mix to each sample. Pipette mix and centrifuge briefly.
  5. Incubate in a thermal cycler (run time: Duration00:30:00 ).
  6. Store at Temperature4 °C for up to Duration18:00:00 or proceed to the next step.
18h 30m
Pre-Amplification Cleaup -- SPRIselect
  1. Add Amount160 µL SPRIselect reagent to each sample and pipette mix thoroughly.
  2. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  3. Centrifuge briefly and place on magnet.
  4. Once solution is clear, remove supernatant.
  5. Add Amount300 µL freshly prepared 80% ethanol. Wait Duration00:00:30 before removing the ethanol.
  6. Add Amount200 µL 80% ethanol. Wait Duration00:00:30 before removing the ethanol.
  7. Centrifuge briefly and place on magnet. Remove any remaining ethanol.
  8. Remove from magnet and immediately add Amount160.5 µL Buffer EB. Pipette mix (pipette set to 150 µl) without introducing bubbles.
  9. Incubate for Duration00:02:00 at TemperatureRoom temperature .
  10. Centrifuge briefly and return to magnet.
  11. After solution clears, transfer Amount160 µL to a new tube strip.
  12. Store at Temperature4 °C for up to Duration72:00:00 or at Temperature-20 °C for long-term storage or proceed to the next step.
3d 0h 8m
Pre-Amplified, SPRI-cleaned Sample Split
  • Move Amount40 µL to a new tube strip for ATAC Library Construction.
  • Move Amount35 µL to a new tube strip for cDNA Amplification.
  • Store the remaining Amount85 µL pre-amplified, SPRI-cleaned product at Temperature-20 °C long term for generating additional libraries.
ATAC Library Construction
ATAC Library Construction
ATAC Sample Index PCR
  1. Bring Sample Index Plate N, Set A to TemperatureRoom temperature .
  2. Thaw, vortex, and centrifuge a tube of SI-Primer B (maintain TemperatureOn ice ).
  3. Centrifuge Amp Mix (maintain TemperatureOn ice ) before adding.
  4. Prepare Sample Index PCR Mix (follow recipe in User Guide).
  5. Add Amount57.5 µL Sample Index PCR Mix to the Amount40 µL aliquot of pre-amplified sample. Pipette mix and centrifuge briefly.
  6. Add Amount2.5 µL of an individual Sample Index N, Set A to each sample. Pipette mix and centrifuge briefly.
  7. Incubate in a thermal cycler to amplify DNA (run time: approximately Duration00:30:00 ).
Note
  • Use Cycle Number Optimization Table (in User Guide) for total number of cycles (based on Targeted Nuclei Recovery).
  • For Targeted Nuclei Recovery of 6,001-10,000 nuclei, perform 7 cycles.

8. Store at Temperature4 °C for up to Duration72:00:00 or proceed to the next step.

3d 0h 30m
ATAC Post Sample Index Double Sided Size Selection -- SPRIselect
  1. Add Amount60 µL SPRIselect reagent (0.6X) to each sample. Pipette mix.
  2. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  3. Place on magnet until solution clears.
  4. Transfer Amount150 µL supernatant to a new tube strip. DO NOT discard the supernatant.
  5. Add Amount95 µL SPRIselect reagent (1.55X) to each sample (supernatant). Pipette mix.
  6. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  7. Place on magnet until solution clears and remove the supernatant.
  8. Add Amount300 µL freshly prepared 80% ethanol to the pellet. Wait Duration00:00:30 before removing ethanol.
  9. Add Amount200 µL 80% ethanol to the pellet. Wait Duration00:00:30 before removing ethanol.
  10. Centrifuge briefly before returning to magnet and removing any residual ethanol.
  11. Remove from the magnet and immediately add Amount20.5 µL Buffer EB. Pipette mix.
  12. Incubate for Duration00:02:00 at TemperatureRoom temperature .
  13. Centrifuge briefly and place on magnet.
  14. Once solution clears, transfer Amount20 µL to a new tube strip.
  15. Store at Temperature4 °C for up to Duration72:00:00 or at Temperature-20 °C for long-term storage.

Note
Post Library Construction QC:
  • Quantify library (e.g. using Qubit dsDNA HS assay).
  • Estimate library size range (e.g. using TapeStation HS D or Bioanalyzer) - expected size range is 170-700 bp, including nucleosome pattern of at least 3 different bands/peaks.


3d 0h 13m
cDNA Amplification PCR & Cleanup
cDNA Amplification PCR & Cleanup
cDNA Amplification
  1. Thaw, vortex, and centrifuge cDNA Primers.
  2. Centrifuge Amp Mix (maintain TemperatureOn ice ) before adding.
  3. Prepare cDNA Amplification Mix TemperatureOn ice (follow recipe in User Guide). Vortex and centrifuge briefly.
  4. Add Amount65 µL cDNA Amplification Reaction Mix to Amount35 µL pre-amplified sample.
  5. Pipette mix 15X (pipette set to 90 µl) and centrifuge briefly.
  6. Incubate in a thermocycler to amplify cDNA (run time: approximately 30-40 minutes).
Note
  • Use Cycle Number Optimization Table (in User Guide) for total number of cycles (based on Targeted Nuclei Recovery).
  • For Targeted Nuclei Recovery >6,000, perform 6 cycles.

7. Store at Temperature4 °C for up to Duration72:00:00 or at Temperature-20 °C for up to 4 weeks, or proceed to the next step.
3d
cDNA Cleanup -- SPRIselect
  1. Add Amount60 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15X (pipette set to 150 µl).
  2. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  3. Place on magnet until solution clears. Remove supernatant
  4. Add Amount200 µL freshly prepared 80% ethanol to the pellet. Wait Duration00:00:30 before removing ethanol.
  5. Repeat step 4 for a total of 2 washes.
  6. Centrifuge briefly before returning to magnet and removing any residual ethanol.
  7. Air dry for Duration00:02:00 (DO NOT exceed 2 minutes as this will decrease elution efficiency).
  8. Remove from the magnet and elute with Amount40.5 µL Qiagen Buffer EB. Pipette mix 15X.
  9. Incubate for Duration00:02:00 at TemperatureRoom temperature .
  10. Place the tube strip on the magnet until the solution clears.
  11. TransferAmount40 µL to a new strip tube.
  12. Store at Temperature4 °C for up to Duration72:00:00 or at Temperature-20 °C for up to 4 weeks, or proceed to the next step.
3d 0h 9m 30s
cDNA QC & Quantification
  1. Quantify library (e.g. using Qubit dsDNA HS assay).
  2. Run TapeStation HS D5000 to obtain concentration and size
  3. Expected size range for amplified cDNA: 400 to 2500 bp
  4. cDNA total yield range: 80 to 1000 ng
Note
QC Cutoff: minimum cDNA total yield of 20 ng.

3' Gene Expression Library Construction
3' Gene Expression Library Construction
Prepare Reagents for use
  1. Pre-cool thermal cycler block to Temperature4 °C prior to preparing the Fragmentation Mix.
  2. Thaw, vortex, and centrifuge Fragmentation Buffer, Adaptor Oligos, Ligation Buffer, SI Primer.
  3. Maintain TemperatureOn ice Fragmentation Enzyme, DNA Ligase, and Amp Mix.
  4. Bring Sample Index Plate TT, Set A to TemperatureRoom temperature .

Fragmentation, End Repair & A-tailing
  1. 1. Prepare Fragmentation Mix TemperatureOn ice (follow recipe in User Guide). Pipette mix and centrifuge briefly.
2. Transfer Amount10 µL purified cDNA sample to a new tube strip TemperatureOn ice .

Note
Note: 10 µl (25%) cDNA sample is sufficient for generating 3' Gene Expression Library. The remaining 30 µl (75%) cDNA sample can be stored at Temperature4 °C for up to Duration72:00:00 or at Temperature-20 °C for up to 4 weeks for generating additional 3' Gene Expression Libraries.


3. Add Amount25 µL Buffer EB to each sample.
4. Add Amount15 µL Fragmentation Mix to each sample.
5. Pipette mix 15X (pipette set to 35 µl) TemperatureOn ice and centrifuge briefly.
6. Transfer to pre-cooled block and “SKIP” hold step to initiate the thermal cycler protocol (run time: approximately Duration00:35:00 ).

35m
Post Fragmentation, End Repair & A-tailing Double Sided Size Selection -- SPRIselect
  1. Add Amount30 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 75 µl).
  2. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  3. Place on magnet and wait until the solution clears. DO NOT discard supernatant.
  4. Transfer Amount75 µL supernatant to a new tube strip.
  5. Add Amount10 µL SPRIselect reagent (0.8X) to each sample and pipette mix 15x (pipette set to 80 µl).
  6. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  7. Place on magnet until the solution clears.
  8. Remove supernatant. DO NOT discard any beads.
  9. Add Amount125 µL freshly prepared 80% ethanol to the pellet. Wait Duration00:00:30 before removing ethanol.
  10. Repeat step 9 for a total of 2 washes.
  11. Centrifuge briefly before returning to magnet and removing any residual ethanol.
  12. Air dry for Duration00:02:00 (DO NOT exceed 2 minutes as this will decrease elution efficiency).
  13. Remove from the magnet and elute with Amount50.5 µL Buffer EB. Pipette mix 15X.
  14. Incubate for Duration00:02:00 at TemperatureRoom temperature .
  15. Place the tube strip on the magnet until the solution clears.
  16. TransferAmount50 µL to a new strip tube.
  17. Store at Temperature4 °C for up to Duration72:00:00 or at Temperature-20 °C for up to 4 weeks, or proceed to the next step.
3d 0h 14m 30s
Adaptor Ligation
  1. Prepare Adaptor Ligation Mix TemperatureOn ice (follow recipe in User Guide). Pipette mix and centrifuge briefly.
  2. Add Amount50 µL Adaptor Ligation Mix to Amount50 µL sample. Pipette mix 15X (pipette set to 90 µl). Centrifuge briefly.
  3. Incubate in a thermal cycler to ligate adaptor (run time: Duration00:15:00 ).
15m
Post Ligation Cleanup -- SPRIselect
  1. Add Amount80 µL SPRIselect reagent (0.8X) to each sample. Pipette mix 15X (pipette set to 150 µl).
  2. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  3. Place on magnet and wait until the solution clears. Remove supernatant.
  4. Add Amount200 µL freshly prepared 80% ethanol to the pellet. Wait Duration00:00:30 before removing ethanol.
  5. Repeat step 4 for a total of 2 washes.
  6. Centrifuge briefly before returning to magnet and removing any residual ethanol.
  7. Air dry for Duration00:02:00 (DO NOT exceed 2 minutes as this will decrease elution efficiency).
  8. Remove from the magnet and elute with Amount30.5 µL Buffer EB. Pipette mix 15X.
  9. Incubate for Duration00:02:00 at TemperatureRoom temperature .
  10. Place the tube strip on the magnet until the solution clears.
  11. TransferAmount30 µL to a new strip tube.
9m 30s
Sample Index PCR
  1. Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run.
  2. Add Amount50 µL Amp Mix to Amount30 µL sample.
  3. Add Amount20 µL of an individual Dual Index TT Set A to each tube and record assignment.
  4. Pipette mix 5X (pipette set to 90 µl) and centrifuge briefly.
  5. Incubate in a thermal cycler to incorporate sample indices (run time: approximately 25-40 minutes).
Note
  • Use Recommended Cycle Numbers Table (in User Guide) for total number of cycles; recommended number of cycles is based on cDNA input.

6. Store at Temperature4 °C for up to Duration72:00:00 or proceed to the next step.

Post Sample Index PCR Double Sided Size Selection -- SPRIselect
  1. Add Amount60 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15X (pipette set to 150 µl).
  2. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  3. Place on magnet until solution clears. DO NOT discard supernatant.
  4. Transfer Amount150 µL supernatant to a new tube strip tube.
  5. Add Amount20 µL SPRIselect reagent (0.8X) to each sample and pipette mix 15x (pipette set to 150 µl).
  6. Incubate for Duration00:05:00 at TemperatureRoom temperature .
  7. Place on magnet and remove supernatant. DO NOT discard any beads.
  8. Add Amount200 µL freshly prepared 80% ethanol to the pellet. Wait Duration00:00:30 before removing ethanol.
  9. Repeat step 8 for a total of 2 washes.
  10. Centrifuge briefly before returning to magnet and removing any residual ethanol.
  11. Air dry for Duration00:02:00 (DO NOT exceed 2 minutes as this will decrease elution efficiency).
  12. Remove from the magnet and elute with Amount35.5 µL Buffer EB. Pipette mix 15X.
  13. Incubate for Duration00:02:00 at TemperatureRoom temperature .
  14. Place the tube strip on the magnet until the solution clears.
  15. TransferAmount35 µL to a new strip tube.
  16. Store at Temperature4 °C for up to Duration72:00:00 or at Temperature-20 °C for long-term storage.
Note
QC Post Library Construction:
  • Quantify Library (e.g. using Qubit dsDNA HS Assay)
  • Estimate Library size range (e.g. using TapeStation HS D1000 or BioAnalyzer) - expected size range of 300 to 800 bp, average: 475 bp.

3d 0h 14m 30s
ATAC Library Sequencing
ATAC Library Sequencing
(Optional) MiSeq Sequencing - QC for estimation of library quality and number of nuclei captured
1. Paired End, Single Indexing
a. Read 1: 50 cycles
b. i7 Index: 8 cycles
c. i5 Index: 24 cycles
d. Read 2: 50 cycles
2. Library Loading
a. 10X recommended Loading concentration: 10 pM
b. Optional: 1% PhiX
3. Output
a. 22-25 million reads
NovaSeq Sequencing (target - 50,000-100,000 reads per nucleus)
1. Paired End, Single Indexing
a. Read 1: 50 cycles
b. i7 Index: 8 cycles
c. i5 Index: 24 cycles
d. Read 2: 50 cycles
2. Library Loading
a. 10X recommended Loading concentration: 300 pM
b. Optional: 1% PhiX
3. Output
a. SP: 650–800 million reads
b. S1: 1.3–1.6 billion reads
c. S2: 3.3 –4.1 billion reads
d. S4: 8-10 billion reads
Gene Expression Library Sequencing
Gene Expression Library Sequencing
(Optional) MiSeq Sequencing - QC for estimation of library quality and number of nuclei captured
1. Paired End, Single Indexing
a. Read 1: 28 cycles
b. i7 Index: 10 cycles
c. i5 Index: 10 cycles
d. Read 2: 91 cycles
2. Library Loading
a. 10X recommended Loading concentration: 11 pM
b. Optional: 1% PhiX
3. Output
a. 22-25 million reads
NovaSeq Sequencing (target - 25,000-50,000 reads per nucleus)
1. Paired End, Single Indexing
a. Read 1: 28 cycles
b. i7 Index: 10 cycles
c. i5 Index: 10 cycles
d. Read 2: 91 cycles
2. Library Loading
a. 10X recommended Loading concentration: 300 pM
b. Optional: 1% PhiX
3. Output
a. SP: 650–800 million reads
b. S1: 1.3–1.6 billion reads
c. S2: 3.3 –4.1 billion reads
d. S4: 8-10 billion reads
Cell Ranger Arc Mapping and Analysis Pipeline
Cell Ranger Arc Mapping and Analysis Pipeline
a. Generate Sample Sheet
Note
Use sample sheet generator provided by 10X Genomics to generate a "SampleSheet.csv":Sample Sheet Generator

b. Generate fastq files
Note
Use cellranger-arc mkfastq command.
c. Generate the libraries CSV file which lists all of the fastqs to be analyzed together. There should be a header, "fastqs,sample,library_type", followed by one row per set of fastqs. The first comma separated value is the path to the directory containing the fastq files, the second value is the sample name used for each set of fastqs, the last value specifies whether the fastq files are for "Gene Expression" or "Chromatin Accessibility" analysis.
Note
Example of a CSV file:

fastqs,sample,library_type
/home/jdoe/runs/HNGEXSQXXX/outs/fastq_path,example,Gene Expression /home/jdoe/runs/HNATACSQXX/outs/fastq_path,example,Chromatin Accessibility

d. Run cellranger-arc count for each sample.

Note
Cellranger-arc counts intronic reads by default.