SenNet URMC 10X Genomics Single-Nucleus RNA-Sequencing for Transcriptomic Profiling

Prepare nuclei according to the protocol "Isolation of single nuclei from solid tissues" steps 1-14. dx.doi.org/10.17504/protocols.io.bh26j8he

Resuspend nuclei in 100µL to 1mL of PBS + 0.1% RNase Inhibitor (volume depends on target concentration)

Perform manual trypan blue nuclear exclusion counts on a hemacytometer.

Check nuclei integrity by light microscope concurrent with manual trypan exclusion counting. 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 700-1200 nuclei per uL range in 50µLtotal volume using the dilution guide

Prepare Reagents for use

1. Equilibrate gel beads to room temperature for 0h 30m 0s.
2. Thaw, vortex, and centrifuge RT Reagent, Template Switch Oligo, and Reducing Agent B. Verify no precipitate in Reducing Agent B.
3. Centrifuge RT Enzyme C before adding to Master Mix.

Prepare Master Mix

1. Prepare on ice. Pipette mix 15x and centrifuge briefly.
2. Add 31.9µL Master Mix to each tube of a PCR 8-tube strip on ice.

Load Chromium Single Cell G Chip

1. Assemble Next GEM Chromium Chip G 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. 75µL into unused wells in Row 1

   2. 40µL into unused wells in Row 2

   3. 45µL into unused wells in Row 3

DO NOT add 50% glycerol solution to the bottom row of NO FILL wells. DO NOT use any substitute for 50% glycerol solution.

5. Use Cell Suspension Volume Calculator Table (see step 1.2 of Chromium Next GEM Single Cell 3' v3.1 protocol) to add the appropriate volume of nuclease-free water to Master Mix already in PCR 8-tube strip. Add corresponding volume of gently pipette-mixed single nuclei suspension to Master Mix. Total of 75µL in each tube. Gently pipette mix, avoid introducing bubbles. DO NOT add nuclease free-water directly to single cell suspension, add instead to Master Mix.

6. Nuclei Loading

   1. Minimum: load 800 nuclei → target recovery 500 nuclei

   2. Maximum: load 1600 nuclei → target recovery 10000 nuclei

7. Load70µL Master Mix + Nuclei Suspension into the bottom center of each well in Row 1 without introducing bubbles.

8. Snap the Gel Bead strip into a 10X Vortex Adapter. Vortex 0h 0m 30s. Remove the Gel Bead strip and flick sharply downward to ensure maximum recovery. Confirm there are no bubbles at the bottom of the tubes and liquid levels look even.

9. Load 50µL gel beads into wells of Row 2. Only puncture the foil seal for gel bead tubes being used. Dispense slowly and without introducing bubbles.

10. Load45µL partitioning oil into each Row 3 by pipetting two aliquots of 140µL.

11. 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 chip immediately after loading the partitioning oil.

Run the Chromium Controller

1. Press the eject button on the controller to eject 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. Firmware Version 4.0 or higher is required in the Chromium Controller or the Chromium Single Cell Controller used for this protocol.

Transfer GEMs

1. Chill strip tubes 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 1-2. Abnormally high volume in any well indicates a clog.

5. Slowly aspirate 100 uL GEMs from the lowest points of the Recovery Wells without creating a seal between the pipette tips and the wells.

   Note

   Inspect the GEMs in the pipette tip. 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.

If multiple chips are run back-to-back, cap/cover the GEM-containing tube strip and place on ice for no more than 1 h.

GEM-RT Incubation

1. Incubate in a thermocycler to complete reverse transcription (53°C 0h 45m 0s , 85°C 0h 5m 0s , 4°C HOLD).
2. Store at 4°C for up to 72h 0m 0sor at -20°C for up to a week. Or proceed to the next step.

Post GEM-RT Cleanup & cDNA Amplification - Prepare Reagents for use (See 10X User Guide for details)

1. Thaw, vortex, and centrifuge a tube of Reducing Agent B and cDNA primers.
2. Maintain Amp Mix on ice after vortex and centrifuge
3. Thaw Cleanup Buffer at 65°C for 0h 10m 0s with shaking at max rpm then cool to room temperature. Verify no visible crystals. Cool to RT.

Post GEM RT-Cleanup --Dynabead

1. Add125µL Recovery Agent to each sample at room temperature. DO NOT MIX. Wait 0h 2m 0s.

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 (See 10X User Guide for detail)

4. Vortex and briefly centrifuge mixture. Add 200µL to each sample. Pipette 10x to mix.

5. Incubate at room temperature for 0h 10m 0s; pipette mix again 0h 5m 0s into incubation.

6. Prepare Elution Solution I (See User Guide for Detail). Vortex and centrifuge briefly.

7. Place sample on magnetic separator-HIGH until solution clears. Remove the supernatant (acqueous phase and Recovery Agent). Add 300µL freshly prepared 80% ethanol. Wait 30 sec. Remove Ethanol. Repeat.Remove the ethanol.

8. Centrifuge briefly. Place on the magnet•Low. Remove remaining ethanol. Air dry for 1 min.

9. Remove from the magnet. Immediately add 35.5 µl Elution Solution I (prepared in step above). Pipette mix (pipette set to 30 µl) without introducing bubbles.

10. Incubate 2 min at room temperature.

11. Place on the magnet•Low until the solution clears. Transfer 35 µl sample to a new tube strip.

cDNA Amplification

1. Prepare cDNA Amplification Mix on ice (see 10x User Guide for details). Vortex and centrifuge briefly.
2. Add 65µL cDNA Amplification Reaction Mix to 35µL sample.
3. Pipette mix 15x to mix and centrifuge briefly.
4. Incubate in a thermocycler to amplify cDNA.
   Note

   Use cycle number optimization table in 10x User Guide) for total number of cyclesWhen using nuclei, increase the optimized number of cycles by one cycleMaximum: load 1600 nuclei - perform 12 cycles

cDNA Cleanup --SPRIselect

1. Perform a 0.6X beads purification with SPRIselect reagent (60µL )
2. Incubate x 5 minutes at RT.
3. Place on magent-HIGH until solution clears. Remove supernatent.
4. Add 200µL freshly prepared 80% ethanol, wait 30 sec and remove ethanol. Repeat.
5. Centrifuge briefly. Place on magnet-LOW. Add 40.5µL Qiagen Buffer EB. Pipette mix x 15x. Incubate 0h 2m 0s RT.
6. Place on magent-HIGH until solution clears. Transfer40µL to a new strip tube.
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 QC & Quantification

1. Run TapeStation to obtain concentration and size
2. Expected size range for amplified cDNA: 400 to 2500 bp
3. cDNA total yield range: 80 to 1000 ng
   Note

   QC Cutoff: Minimum cDNA total yield of 80 ng

Prepare Reagents for use

1. Thaw, vortex, and centrifuge Fragmentation Buffer, Adaptor Oligos, Ligation Buffer, SI Primer. Verify no precipitate.
2. Maintain on ice Fragmentation Enzyme, DNA Ligase, and Amp Mix.

Fragmentation, End Repair & A-tailing

1. Prepare Fragmentation Mix on ice (See User Manual for volume details). Pipette mix and centrifuge briefly.

2. Transfer 10µL purified cDNA to a new strip tube

3. Add 25µL Buffer EB to each sample

4. Add 15µL Fragmentation Mix to each sample

5. Pipette mix, centrifuge briefly, and transfer to pre-cooled block. “SKIP” hold step to initiate the protocol

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

1. Vortex to resuspend the SPRIselect reagent. Add 30µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x.
2. Incubate 0h 5m 0s at room temperature.
3. Centrifuge briefly.
4. Place sample on magnetic separator-HIGH until solution clears. DO NOT discard supernatant.
5. Transfer 75µL supernatant to a new tube strip tube.
6. Vortex to resuspend the SPRIselect reagent. Add 10µL SPRIselect reagent (0.8X) to each transferred supernatent and pipette mix 15x.
7. Incubate 0h 5m 0s RT
8. Place sample on magnetic separator-HIGH until solution clears.
9. Remove 80µL supernatant. DO NOT discard any beads.
10. Wash twice with 125µL freshly prepared 80% ethanol.
11. Centrifuge briefly. Place on magent-LOW until solution clears. Remove remaining ethanol, do not overdry.
12. Remove from magnet. Add 50.5µL Qiagen Buffer EB, pipette mix x 15. Incubate x 0h 2m 0s RT.
13. Place on magent-HIGH until clear. Transfer 50µL to a new strip tube.

Adaptor Ligation

1. Prepare Adaptor Ligation Mix on ice. Pipette mix and centrifuge briefly.
2. Add 50µL Adaptor Ligation Mix to sample. Pipette mix 15x. Centrifuge briefly.
3. Incubate in a thermocycler to ligate adaptor. (20°C x 0h 15m 0s , 4°C Hold)

Post Ligation Cleanup --SPRIselect

1. Perform a 0.8X beads purification with SPRIselect reagent (add 80µL reagent to each sample, pipette-mix x 15x, incubate 5 min at RT)
2. Place on magnet-HIGH until solution clears, remove supernatant. Wash twice with freshly prepared 80% ethanol (add 200µL, wait 30 sec, remove ethanol and repeat)
3. Centrifuge briefly. Place on magent-LOW. Remove remaining ethanol and air dry up to 2 min.
4. Remove from magnet. To elute, add 30.5µL Qiagen Buffer EB. Pipette mix x 15. Incubate 2 min at RT. Place on magent-LOW until solution clears.
5. Transfer 30µL 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. Prepare Sample Index PCR Mix on ice. Pipette mix and centrifuge briefly.

3. Add 50µL Amp Mix to the 30µL sample already in the new strip tube.

4. Add 20µL of an individual Dual Index TT Set A to each sample. Record well assignment. Pipette-mix x5 and centrifuge briefly.

5. Incubate in a thermocycler to incorporate sample indices.

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. Vortex to resuspend the SPRIselect reagent. Add 60µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x.
2. Incubate 0h 5m 0s at room temperature.
3. Centrifuge briefly
4. Place sample on magnetic separator. DO NOT discard supernatant.
5. Transfer 150µL supernatant to a new tube strip tube.
6. Vortex to resuspend the SPRIselect reagent. Add 20µL SPRIselect reagent (0.8X) to each sample and pipette mix 15x.
7. Incubate 0h 5m 0s at room temperature.
8. Centrifuge briefly
9. Place sample on magnetic separator-HIGH until solution clears.
10. Remove 165µL supernatant. DO NOT discard any beads.
11. With tube still on magnet, add 200µLfreshly prepared 80% ethanol to pellet. Wait 30 sec and then remove ethanol.
12. Repeat Step 11 for total of 2 washes then centrifuge briefly. Place on magnet-LOW. Remove remaining ethanol.
13. Remove from magnet. Add 35.5µL Qiagen Buffer EB. Pipette-mix x 15)
14. Incubate 2 min at RT.
15. Place on magnet-LOW until solution clears then transfer 35µL 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 or BioAnalyzer) - expected size range of 300 to 800 bp, average: 475 bp If additional peaks below 200 bp are observed, see User Guide v 3.1 for recommendations

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: 8 cycles

   3. i5 Index: 0 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: 8 cycles

   3. i5 Index: 0 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

Generate pre-mRNA reference

b. Generate Sample Sheet

c. Generate fastq files

d. Run count for each sample