10X Genomics Single-Nucleus Multiome (RNA + ATAC) Assay for Profiling Adult Human Tissues

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. 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 50µL to 100µL 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 5µLtotal volume using the dilution guide

Prepare Reagents for use

1. Thaw, vortex, and centrifuge ATAC Buffer B.
2. Centrifuge ATAC Enzyme B (maintain On ice) before adding to Transposition Mix.

Prepare Transposition Mix.

1. Prepare On ice. Pipette mix 10X and centrifuge briefly.
2. Add 10µL Transposition Mix to each tube of a PCR 8-tube strip On ice.
3. Calculate the volume of Nuclei Stock and Diluted Nuclei Buffer for a total volume of 5µL 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 37°C for 1h 0m 0s in a thermal cycler with lid set at 50°C for transposition and addition of adapter sequences, which are added to the ends of the DNA fragments.
2. Proceed immediately to next step.

Prepare Reagents for use

1. Equilibrate gel beads to Room temperature for 0h 30m 0s.
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 On ice) before adding to Master Mix.

Prepare Master Mix

1. PrepareOn ice. Pipette mix 10X and centrifuge briefly.
2. Keep On ice.
3. Add 60.0µL Master Mix to each tube containing Transposed Nuclei On 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)

   1. 70µL into unused wells in Row 1

   2. 50µL into unused wells in Row 2

   3. 45µL into unused wells in Row 3

5. Add 60.0µL Master Mix to each tube containing Transposed Nuclei on ice. Gently pipette mix 5X.

6. Load 70µL 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 0h 0m 30s. Remove the Gel Bead strip and centrifuge for ~ 0h 0m 5s. Confirm there are no bubbles at the bottom of the tubes and liquid levels look even.

8. Load 50µL Gel Beads in Row 2. Dispense slowly and without introducing bubbles. Wait 0h 0m 30s.

9. Load 40µL 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.

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 (~0h 18m 0s), the Controller will chime. Proceed immediately to the next step.

Transfer GEMs

1. Chill a PCR 8-tube strip On 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 (~0h 0m 20s) GEMs into the tube strip On ice with the pipette tips against the sidewalls of the wells.

GEM Incubation

1. Incubate in a thermocycler for 1h 15m 0s to complete reverse transcription.
2. During GEM incubation, equilibrate Quenching Agent to Room temperature.
3. Upon completion of GEM incubation, proceed immediately to next step.

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 -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 65°C for 0h 10m 0s 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 125µL 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 125µL 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 200µL Dynabeads Cleanup Mix to each sample. Pipette 10X to mix (pipette set to 200 µl).
5. Incubate at room temperature for 0h 10m 0s 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 300µL of freshly prepared 80% ethanol. Wait 0h 0m 30s before removing ethanol.
9. Add 200µL of freshly prepared 80% ethanol. Wait 0h 0m 30s before removing ethanol.
10. Remove from magnet and immediately add 50µL Elution Solution 1 to avoid clumping. Pipette mix (pipette set to 50 µl) without introducing bubbles.
11. Incubate 0h 1m 0s at Room temperature .
12. Centrifuge briefly and move back to magnet.
13. After solution clears, transfer 50µL to a new tube strip.

Post GEM Incubation Cleanup -- SPRIselect

1. Add 90µL SPRIselect reagent to each sample and pipette mix thoroughly.
2. Incubate 0h 5m 0s at Room temperature.
3. Centrifuge briefly and place on magnet until solution clears. Remove supernatant.
4. Add 200µL freshly prepared 80% ethanol to pellet and wait 0h 0m 30s 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 46.5µL Buffer EB. Pipette mix (pipette set to 45 µl) without introducing bubbles.
9. Incubate 0h 2m 0s at Room temperature.
10. Centrifuge briefly and return to magnet.
11. After solution clears, transfer 46µL to a new tube strip.

Pre-Amplification PCR

1. Thaw, vortex, and centrifuge Pre-Amp Primers.
2. Centrifuge Pre-Amp Mix (maintain On ice) before adding.
3. Prepare Pre-Amplification Mix On ice (follow recipe in User Guide). Pipette mix 10X and centrifuge briefly.
4. Add 54µL Pre-Amplification Mix to each sample. Pipette mix and centrifuge briefly.
5. Incubate in a thermal cycler (run time: 0h 30m 0s).
6. Store at 4°C for up to 18h 0m 0s or proceed to the next step.

Pre-Amplification Cleaup -- SPRIselect

1. Add 160µL SPRIselect reagent to each sample and pipette mix thoroughly.
2. Incubate for 0h 5m 0s at Room temperature.
3. Centrifuge briefly and place on magnet.
4. Once solution is clear, remove supernatant.
5. Add 300µL freshly prepared 80% ethanol. Wait 0h 0m 30s before removing the ethanol.
6. Add 200µL 80% ethanol. Wait 0h 0m 30s before removing the ethanol.
7. Centrifuge briefly and place on magnet. Remove any remaining ethanol.
8. Remove from magnet and immediately add 160.5µL Buffer EB. Pipette mix (pipette set to 150 µl) without introducing bubbles.
9. Incubate for 0h 2m 0s at Room temperature.
10. Centrifuge briefly and return to magnet.
11. After solution clears, transfer 160µL to a new tube strip.
12. Store at 4°C for up to 72h 0m 0s or at -20°C for long-term storage or proceed to the next step.

Pre-Amplified, SPRI-cleaned Sample Split

• Move 40µL to a new tube strip for ATAC Library Construction.
• Move 35µL to a new tube strip for cDNA Amplification.
• Store the remaining 85µL pre-amplified, SPRI-cleaned product at -20°C long term for generating additional libraries.

ATAC Sample Index PCR

1. Bring Sample Index Plate N, Set A to Room temperature.

2. Thaw, vortex, and centrifuge a tube of SI-Primer B (maintain On ice).

3. Centrifuge Amp Mix (maintain On ice) before adding.

4. Prepare Sample Index PCR Mix (follow recipe in User Guide).

5. Add 57.5µL Sample Index PCR Mix to the 40µL aliquot of pre-amplified sample. Pipette mix and centrifuge briefly.

6. Add 2.5µL 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 0h 30m 0s).

   Note

   Use Cycle Number Optimization Table (in Use Cycle Number Optimization Table (in User Guide) for total number of cycles (based on Targeted Nuclei Recovery).) 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 4°C for up to 72h 0m 0s or proceed to the next step.

ATAC Post Sample Index Double Sided Size Selection -- SPRIselect

1. Add 60µL SPRIselect reagent (0.6X) to each sample. Pipette mix.
2. Incubate for 0h 5m 0s at Room temperature.
3. Place on magnet until solution clears.
4. Transfer 150µL supernatant to a new tube strip. DO NOT discard the supernatant.
5. Add 95µL SPRIselect reagent (1.55X) to each sample (supernatant). Pipette mix.
6. Incubate for 0h 5m 0s at Room temperature.
7. Place on magnet until solution clears and remove the supernatant.
8. Add 300µL freshly prepared 80% ethanol to the pellet. Wait 0h 0m 30s before removing ethanol.
9. Add 200µL 80% ethanol to the pellet. Wait 0h 0m 30s before removing ethanol.
10. Centrifuge briefly before returning to magnet and removing any residual ethanol.
11. Remove from the magnet and immediately add 20.5µL Buffer EB. Pipette mix.
12. Incubate for 0h 2m 0s at Room temperature.
13. Centrifuge briefly and place on magnet.
14. Once solution clears, transfer 20µL to a new tube strip.
15. Store at 4°C for up to 72h 0m 0s or at -20°C for long-term storage.

cDNA Amplification

1. Thaw, vortex, and centrifuge cDNA Primers.

2. Centrifuge Amp Mix (maintain On ice) before adding.

3. Prepare cDNA Amplification Mix On ice (follow recipe in User Guide). Vortex and centrifuge briefly.

4. Add 65µL cDNA Amplification Reaction Mix to 35µL 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 Use Cycle Number Optimization Table (in User Guide) for total number of cycles (based on Targeted Nuclei Recovery).) for total number of cycles (based on Targeted Nuclei Recovery).For Targeted Nuclei Recovery >6,000, perform 6 cycles.

7. Store at 4°C for up to 72h 0m 0s or at -20°C for up to 4 weeks, or proceed to the next step.

cDNA Cleanup -- SPRIselect

1. Add 60µL SPRIselect reagent (0.6X) to each sample and pipette mix 15X (pipette set to 150 µl).
2. Incubate for 0h 5m 0s at Room temperature.
3. Place on magnet until solution clears. Remove supernatant
4. Add 200µL freshly prepared 80% ethanol to the pellet. Wait 0h 0m 30s 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 0h 2m 0s (DO NOT exceed 2 minutes as this will decrease elution efficiency).
8. Remove from the magnet and elute with 40.5µL Qiagen Buffer EB. Pipette mix 15X.
9. Incubate for 0h 2m 0s at Room temperature.
10. Place the tube strip on the magnet until the solution clears.
11. Transfer40µL to a new strip tube.
12. Store at 4°C for up to 72h 0m 0s or at -20°C for up to 4 weeks, or proceed to the next step.

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.

Prepare Reagents for use

1. Pre-cool thermal cycler block to 4°C prior to preparing the Fragmentation Mix.
2. Thaw, vortex, and centrifuge Fragmentation Buffer, Adaptor Oligos, Ligation Buffer, SI Primer.
3. Maintain On ice Fragmentation Enzyme, DNA Ligase, and Amp Mix.
4. Bring Sample Index Plate TT, Set A to Room temperature.

Fragmentation, End Repair & A-tailing

1. 1. Prepare Fragmentation Mix On ice (follow recipe in User Guide). Pipette mix and centrifuge briefly.
2. Transfer 10µL purified cDNA sample to a new tube strip -20On ice.

3. Add 25µL Buffer EB to each sample.

4. Add 15µL Fragmentation Mix to each sample.

5. Pipette mix 15X (pipette set to 35 µl) -20On ice and centrifuge briefly.

6. Transfer to pre-cooled block and “SKIP” hold step to initiate the thermal cycler protocol (run time: approximately 0h 35m 0s).

Post Fragmentation, End Repair & A-tailing Double Sided Size Selection -- SPRIselect

1. Add 30µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 75 µl).
2. Incubate for 0h 5m 0s at -20Room temperature.
3. Place on magnet and wait until the solution clears. DO NOT discard supernatant.
4. Transfer 75µL supernatant to a new tube strip.
5. Add 10µL SPRIselect reagent (0.8X) to each sample and pipette mix 15x (pipette set to 80 µl).
6. Incubate for 0h 5m 0s at -20Room temperature.
7. Place on magnet until the solution clears.
8. Remove supernatant. DO NOT discard any beads.
9. Add 125µL freshly prepared 80% ethanol to the pellet. Wait 0h 0m 30s 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 0h 2m 0s (DO NOT exceed 2 minutes as this will decrease elution efficiency).
13. Remove from the magnet and elute with 50.5µL Buffer EB. Pipette mix 15X.
14. Incubate for 0h 2m 0s at Room temperature.
15. Place the tube strip on the magnet until the solution clears.
16. Transfer50µL to a new strip tube.
17. Store at 4°C for up to 72h 0m 0s or at -20°C for up to 4 weeks, or proceed to the next step.

Adaptor Ligation

1. Prepare Adaptor Ligation Mix -20On ice (follow recipe in User Guide). Pipette mix and centrifuge briefly.
2. Add 50µL Adaptor Ligation Mix to 50µL sample. Pipette mix 15X (pipette set to 90 µl). Centrifuge briefly.
3. Incubate in a thermal cycler to ligate adaptor (run time: 0h 15m 0s).

Post Ligation Cleanup -- SPRIselect

1. Add 80µL SPRIselect reagent (0.8X) to each sample. Pipette mix 15X (pipette set to 150 µl).
2. Incubate for 0h 5m 0s at -20Room temperature.
3. Place on magnet and wait until the solution clears. Remove supernatant.
4. Add 200µL freshly prepared 80% ethanol to the pellet. Wait 0h 0m 30s 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 0h 2m 0s (DO NOT exceed 2 minutes as this will decrease elution efficiency).
8. Remove from the magnet and elute with 30.5µL Buffer EB. Pipette mix 15X.
9. Incubate for 0h 2m 0s at Room temperature.
10. Place the tube strip on the magnet until the solution clears.
11. Transfer30µL to a new strip tube.

Sample Index PCR

1. Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run.

2. Add 50µL Amp Mix to 30µL sample.

3. Add 20µL 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 Use Recommended Cycle Numbers Table (in User Guide) for total number of cycles; recommended number of cycles is based on cDNA input.) for total number of cycles; recommended number of cycles is based on cDNA input.

6. Store at 4°C for up to 72h 0m 0s or proceed to the next step.

Post Sample Index PCR Double Sided Size Selection -- SPRIselect

1. Add 60µL SPRIselect reagent (0.6X) to each sample and pipette mix 15X (pipette set to 150 µl).
2. Incubate for 0h 5m 0s at -20Room temperature.
3. Place on magnet until solution clears. DO NOT discard supernatant.
4. Transfer 150µL supernatant to a new tube strip tube.
5. Add 20µL SPRIselect reagent (0.8X) to each sample and pipette mix 15x (pipette set to 150 µl).
6. Incubate for 0h 5m 0s at -20Room temperature.
7. Place on magnet and remove supernatant. DO NOT discard any beads.
8. Add 200µL freshly prepared 80% ethanol to the pellet. Wait 0h 0m 30s 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 0h 2m 0s (DO NOT exceed 2 minutes as this will decrease elution efficiency).
12. Remove from the magnet and elute with 35.5µL Buffer EB. Pipette mix 15X.
13. Incubate for 0h 2m 0s at Room temperature.
14. Place the tube strip on the magnet until the solution clears.
15. Transfer35µL to a new strip tube.
16. Store at 4°C for up to 72h 0m 0s or at -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.

MiSeq Sequencing - QC for estimation of library quality and number of nuclei captured

1. Paired End, Single Indexing

   1. Read 1: 50 cycles

   2. i7 Index: 8 cycles

   3. i5 Index: 24 cycles

   4. Read 2: 50 cycles

2. Library Loading

   1. 10X recommended Loading concentration: 10 pM

   2. Optional: 1% PhiX

3. Output

   1. 22-25 million reads

NovaSeq Sequencing (target - 50,000-100,000 reads per nucleus)

1. Paired End, Single Indexing

   1. Read 1: 50 cycles

   2. i7 Index: 8 cycles

   3. i5 Index: 24 cycles

   4. Read 2: 50 cycles

2. Library Loading

   1. 10X recommended Loading concentration: 300 pM

   2. Optional: 1% PhiX

3. Output

   1. SP: 650–800 million reads

   2. S1: 1.3–1.6 billion reads

   3. S2: 3.3 –4.1 billion reads

   4. S4: 8-10 billion reads

MiSeq Sequencing - QC for estimation of library quality and number of nuclei captured

1. Paired End, Single Indexing

   1. Read 1: 28 cycles

   2. i7 Index: 10 cycles

   3. i5 Index: 10 cycles

   4. Read 2: 91 cycles

2. Library Loading

   1. 10X recommended Loading concentration: 11 pM

   2. Optional: 1% PhiX

3. Output

   1. 22-25 million reads

NovaSeq Sequencing (target - 25,000-50,000 reads per nucleus)

1. Paired End, Single Indexing

   1. Read 1: 28 cycles

   2. i7 Index: 10 cycles

   3. i5 Index: 10 cycles

   4. Read 2: 91 cycles

2. Library Loading

   1. 10X recommended Loading concentration: 300 pM

   2. Optional: 1% PhiX

3. Output

   1. SP: 650–800 million reads

   2. S1: 1.3–1.6 billion reads

   3. S2: 3.3 –4.1 billion reads

   4. S4: 8-10 billion reads

a. Generate Sample Sheet

b. Generate fastq files

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.

d. Run cellranger-arc count for each sample.