TITLE: 10X Genomics Single-Nucleus Multiome (RNA + ATAC) Assay for Profiling Adult Human Tissues
AUTHORS: Kimberly Conklin, Bo Zhang, Amanda  Knoten, Dinh Diep, Blue  Lake, Sanjay Jain, Kun Zhang

[DESCRIPTION]
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)

[STEPS]
SECTION: Isolate Nuclei
1. 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.
SECTION: Isolate Nuclei
2. Resuspend nuclei in 30 µL to 100 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).

SECTION: Isolate Nuclei
3. Count nuclei (e.g. BioRad T20 Cell Counter) .
SECTION: Isolate Nuclei
4. 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. 
SECTION: Isolate Nuclei
5. Dilute nuclei stock to be in the 1,000-3,200 nuclei per µL range in 5 µLtotal volume using the dilution guide
 



SECTION: Transposition
6. Prepare Reagents for use
Thaw, vortex, and centrifuge ATAC Buffer B.
Centrifuge ATAC Enzyme B (maintain on ice) before adding to Transposition Mix.

SECTION: Transposition
7. Prepare Transposition Mix.
Prepare on ice. Pipette mix 10X and centrifuge briefly.
Add 10 µL Transposition Mix to each tube of a PCR 8-tube strip on ice.
Calculate the volume of Nuclei Stock and Diluted Nuclei Buffer for a total volume of 5 µL using the Nuclei Concentration Guidelines.
Add the calculated volume of Diluted Nuclei Buffer to the Transposition Mix, pipette mix, and centrifuge briefly.
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.


SECTION: Transposition
8. Isothermal Incubation
Incubate at 37 °C  for 60 min 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.
Proceed immediately to next step.



SECTION: GEM Generation and Barcoding
9. Prepare Reagents for use 
Equilibrate gel beads to Room temperature for 30 min. 
Thaw, vortex, and centrifuge Barcoding Reagent Mix, Template Switch Oligo, and Reducing Agent B. Verify no precipitate in Reducing Agent B. 
Centrifuge Barcoding Enzyme Mix (maintain on ice) before adding to Master Mix.


SECTION: GEM Generation and Barcoding
10. Prepare Master Mix
Prepareon ice. Pipette mix 10X and centrifuge briefly. 
Keep on ice.
Add 60.0 µL Master Mix to each tube containing Transposed Nuclei on ice.



SECTION: GEM Generation and Barcoding
11. 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.	70 µL into unused wells in Row 1 
          b.  50 µL into unused wells in Row 2 
          c.	45 µL into unused wells in Row 3 
5.  Add 60.0 µL Master Mix to each tube containing Transposed Nuclei on ice.  Gently pipette mix 5X.
6.  Load 70 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 30 s. Remove the Gel Bead strip and centrifuge for ~ 5 s.  Confirm there are no bubbles at the bottom of the tubes and liquid levels look even. 
8.	Load 50 µL Gel Beads in Row 2. Dispense slowly and without introducing bubbles. Wait 30 s.
9.	Load 40 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.
SECTION: GEM Generation and Barcoding
12. Run the Chromium Controller
Press the eject button on the Controller to eject the tray.
Place the assembled chip with the gasket in the tray. Press the button to retract the tray.
Confirm the program on screen. Press the play button. 
At completion of the run (~18 min), the Controller will chime. Proceed immediately to the next step.

 


SECTION: GEM Generation and Barcoding
13. Transfer GEMs 
Chill a PCR 8-tube strip on ice. 
Press the eject button to remove the Chip. 
Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees. 
Check the volume in rows 1-3. Abnormally high volume in any well indicates a clog. 
Slowly aspirate 100 µL GEMs from the lowest points of the Recovery Wells without creating a seal between the pipette tips and the wells. 
 

6.  Slowly dispense (~20 s) GEMs into the tube strip on ice with the pipette tips against the sidewalls of the wells.


SECTION: GEM Generation and Barcoding
14. GEM Incubation
Incubate in a thermocycler for 75 min to complete reverse transcription. 
During GEM incubation, equilibrate Quenching Agent to Room temperature.
Upon completion of GEM incubation, proceed immediately to next step.
SECTION: GEM Generation and Barcoding
15. Quenching Reaction
Add 5 µl Quenching Agent to each sample to stop the reaction.
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.
Store at -80 °C  for up to 4 weeks or proceed to the next step.
SECTION: GEM Generation and Barcoding
16. Post GEM Incubation Cleanup -- prepare reagents for use 
Thaw, vortex, and centrifuge a tube of Reducing Agent B.
Thaw Cleanup Buffer at 65 °C for 10 min with shaking at max rpm on a thermomixer, then cool to room temperature before use. Verify there are no visible crystals.
SECTION: GEM Generation and Barcoding
17. Post GEM Incubation Cleanup -- Dynabeads 
Add 125 µL Recovery Agent to each sample at room temperature. DO NOT pipette mix or vortex.  Gently invert tube 10X to mix and centrifuge briefly.
Carefully aspirate 125 µL of pink oil phase from the bottom of the tube. DO NOT aspirate any aqueous sample. 
Prepare Dynabeads Cleanup Mix (follow recipe in User Guide).
Vortex and add 200 µL Dynabeads Cleanup Mix to each sample. Pipette 10X to mix (pipette set to 200 µl).
Incubate at room temperature for 10 min with caps open. 
Prepare Elution Solution I (follow recipe in User Guide). Vortex and centrifuge briefly. 
After the 10-minute incubation period, place the samples on a magnetic separator.  Wait until solution clears before removing the supernatant. 
Add  300 µL  of freshly prepared 80% ethanol.  Wait 30 s before removing ethanol.
Add 200 µL  of freshly prepared 80% ethanol.  Wait 30 s before removing ethanol.
Remove from magnet and immediately add 50 µL  Elution Solution 1 to avoid clumping.  Pipette mix (pipette set to 50 µl) without introducing bubbles.
Incubate 1 min at Room temperature .
Centrifuge briefly and move back to magnet.
After solution clears, transfer 50 µL to a new tube strip.
SECTION: GEM Generation and Barcoding
18. Post GEM Incubation Cleanup -- SPRIselect
Add 90 µL SPRIselect reagent to each sample and pipette mix thoroughly.
Incubate 5 min at Room temperature.
Centrifuge briefly and place on magnet until solution clears.  Remove supernatant.
Add 200 µL freshly prepared 80% ethanol to pellet and wait 30 s before removing ethanol.
Repeat step 4 for a total of 2 washes. 
Centrifuge briefly and place on magnet.
Remove any remaining ethanol (note: residual ethanol can inhibit Pre-Amplification PCR and impact assay performance).
Remove from magnet and immediately add 46.5 µL Buffer EB.  Pipette mix (pipette set to 45 µl) without introducing bubbles.
Incubate 2 min at Room temperature.
Centrifuge briefly and return to magnet.
After solution clears, transfer 46 µL to a new tube strip.
SECTION: Pre-Amplification PCR &amp; Cleanup
19. Pre-Amplification PCR
Thaw, vortex, and centrifuge Pre-Amp Primers.
Centrifuge Pre-Amp Mix (maintain on ice) before adding.
Prepare Pre-Amplification Mix on ice  (follow recipe in User Guide). Pipette mix 10X and centrifuge briefly.
Add 54 µL Pre-Amplification Mix to each sample. Pipette mix and centrifuge briefly.
Incubate in a thermal cycler (run time: 30 min).
Store at 4 °C for up to 1080 min or proceed to the next step.
SECTION: Pre-Amplification PCR &amp; Cleanup
20. Pre-Amplification Cleaup -- SPRIselect
Add 160 µL SPRIselect reagent to each sample and pipette mix thoroughly.
Incubate for 5 min at Room temperature.
Centrifuge briefly and place on magnet.
Once solution is clear, remove supernatant.
Add 300 µL freshly prepared 80% ethanol.  Wait 30 s before removing the ethanol.
Add 200 µL 80% ethanol. Wait 30 s before removing the ethanol.
Centrifuge briefly and place on magnet.  Remove any remaining ethanol.
Remove from magnet and immediately add 160.5 µL Buffer EB.  Pipette mix (pipette set to 150 µl) without introducing bubbles.
Incubate for 2 min at Room temperature.
Centrifuge briefly and return to magnet.
After solution clears, transfer 160 µL to a new tube strip.
Store at 4 °C for up to 4320 min or at -20 °C for long-term storage or proceed to the next step.
SECTION: Pre-Amplification PCR &amp; Cleanup
21. Pre-Amplified, SPRI-cleaned Sample Split
Move 40 µL to a new tube strip for ATAC Library Construction.
Move 35 µL to a new tube strip for cDNA Amplification.
Store the remaining 85 µL pre-amplified, SPRI-cleaned product at -20 °C long term for generating additional libraries.
SECTION: ATAC Library Construction
22. ATAC Sample Index PCR
Bring Sample Index Plate N, Set A to Room temperature.
Thaw, vortex, and centrifuge a tube of SI-Primer B (maintain on ice).
Centrifuge Amp Mix (maintain on ice) before adding.
Prepare Sample Index PCR Mix (follow recipe in User Guide).
Add 57.5 µL Sample Index PCR Mix to the 40 µL aliquot of pre-amplified sample. Pipette mix and centrifuge briefly.
Add 2.5 µL of an individual Sample Index N, Set A to each sample. Pipette mix and centrifuge briefly.
Incubate in a thermal cycler to amplify DNA (run time: approximately 30 min).
 

  8. Store at 4 °C for up to 4320 min or proceed to the next step.

SECTION: ATAC Library Construction
23. ATAC Post Sample Index Double Sided Size Selection -- SPRIselect
Add 60 µL SPRIselect reagent (0.6X) to each sample. Pipette mix.
Incubate for 5 min at Room temperature.
Place on magnet until solution clears.
Transfer 150 µL supernatant to a new tube strip. DO NOT discard the supernatant.
Add 95 µL SPRIselect reagent (1.55X) to each sample (supernatant). Pipette mix.
Incubate for 5 min at Room temperature.
Place on magnet until solution clears and remove the supernatant.
Add 300 µL freshly prepared 80% ethanol to the pellet. Wait 30 s before removing ethanol.
Add 200 µL 80% ethanol to the pellet. Wait 30 s before removing ethanol.
Centrifuge briefly before returning to magnet and removing any residual ethanol.
Remove from the magnet and immediately add 20.5 µL Buffer EB.  Pipette mix.
Incubate for 2 min at Room temperature.
Centrifuge briefly and place on magnet.
Once solution clears, transfer 20 µL to a new tube strip.
Store at 4 °C for up to 4320 min or at -20 °C for long-term storage.

 


SECTION: cDNA Amplification PCR &amp; Cleanup
24. cDNA Amplification 
Thaw, vortex, and centrifuge cDNA Primers.
Centrifuge Amp Mix (maintain on ice) before adding.
Prepare cDNA Amplification Mix on ice (follow recipe in User Guide). Vortex and centrifuge briefly. 
Add 65 µL cDNA Amplification Reaction Mix to 35 µL pre-amplified sample.
Pipette mix 15X (pipette set to 90 µl)  and centrifuge briefly.
Incubate in a thermocycler to amplify cDNA (run time: approximately 30-40 minutes).
 

  7. Store at 4 °C for up to 4320 min or at -20 °C for up to 4 weeks, or proceed to the next step.
SECTION: cDNA Amplification PCR &amp; Cleanup
25. cDNA Cleanup -- SPRIselect 
Add 60 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15X (pipette set to 150 µl).
Incubate for 5 min at Room temperature.
Place on magnet until solution clears. Remove supernatant
Add 200 µL freshly prepared 80% ethanol to the pellet. Wait 30 s before removing ethanol.
Repeat step 4 for a total of 2 washes.
Centrifuge briefly before returning to magnet and removing any residual ethanol.
Air dry for 2 min (DO NOT exceed 2 minutes as this will decrease elution efficiency).
Remove from the magnet and elute with 40.5 µL Qiagen Buffer EB. Pipette mix 15X.
Incubate for 2 min at Room temperature.
Place the tube strip on the magnet until the solution clears.
Transfer40 µL to a new strip tube.
Store at 4 °C for up to 4320 min or at -20 °C for up to 4 weeks, or proceed to the next step.
SECTION: cDNA Amplification PCR &amp; Cleanup
26. cDNA QC & Quantification
Quantify library (e.g. using Qubit dsDNA HS assay).
Run TapeStation HS D5000 to obtain concentration and size 
Expected size range for amplified cDNA: 400 to 2500 bp 
cDNA total yield range: 80 to 1000 ng
 

SECTION: 3&#x27; Gene Expression Library Construction
27. Prepare Reagents for use 
Pre-cool thermal cycler block to 4 °C  prior to preparing the Fragmentation Mix.
Thaw, vortex, and centrifuge Fragmentation Buffer, Adaptor Oligos, Ligation Buffer, SI Primer. 
Maintain on ice Fragmentation Enzyme, DNA Ligase, and Amp Mix.
Bring Sample Index Plate TT, Set A to Room temperature.

SECTION: 3&#x27; Gene Expression Library Construction
28. Fragmentation, End Repair & A-tailing
1.  Prepare Fragmentation Mix on ice (follow recipe in User Guide). Pipette mix and centrifuge briefly. 
2.  Transfer 10 µL purified cDNA sample to a new tube strip on ice.

 

3.  Add 25 µL Buffer EB to each sample.
4.  Add 15 µL Fragmentation Mix to each sample.
5.  Pipette mix 15X (pipette set to 35 µl) on ice and centrifuge briefly.
6.  Transfer to pre-cooled block and “SKIP” hold step to initiate the thermal cycler protocol (run time: approximately 35 min).

SECTION: 3&#x27; Gene Expression Library Construction
29. Post Fragmentation, End Repair & A-tailing Double Sided Size Selection -- SPRIselect 
Add 30 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 75 µl). 
Incubate for 5 min at -20 Room temperature.
Place on magnet and wait until the solution clears. DO NOT discard supernatant. 
Transfer 75 µL supernatant to a new tube strip.  
Add 10 µL SPRIselect reagent (0.8X) to each sample and pipette mix 15x (pipette set to 80 µl). 
Incubate for 5 min at -20 Room temperature.
Place on magnet until the solution clears. 
Remove supernatant. DO NOT discard any beads. 
Add 125 µL freshly prepared 80% ethanol to the pellet. Wait 30 s before removing ethanol.
Repeat step 9 for a total of 2 washes.
Centrifuge briefly before returning to magnet and removing any residual ethanol.
Air dry for 2 min (DO NOT exceed 2 minutes as this will decrease elution efficiency).
Remove from the magnet and elute with 50.5 µL Buffer EB. Pipette mix 15X.
Incubate for 2 min at Room temperature.
Place the tube strip on the magnet until the solution clears.
Transfer50 µL to a new strip tube.
Store at 4 °C for up to 4320 min or at -20 °C for up to 4 weeks, or proceed to the next step.
SECTION: 3&#x27; Gene Expression Library Construction
30. Adaptor Ligation
Prepare Adaptor Ligation Mix on ice (follow recipe in User Guide). Pipette mix and centrifuge briefly.
Add 50 µL Adaptor Ligation Mix to 50 µL sample. Pipette mix 15X (pipette set to 90 µl). Centrifuge briefly.
Incubate in a thermal cycler to ligate adaptor (run time: 15 min).
SECTION: 3&#x27; Gene Expression Library Construction
31. Post Ligation Cleanup -- SPRIselect 
Add 80 µL SPRIselect reagent (0.8X) to each sample.  Pipette mix 15X (pipette set to 150 µl).
Incubate for 5 min at -20 Room temperature.
Place on magnet and wait until the solution clears.  Remove supernatant.
Add 200 µL freshly prepared 80% ethanol to the pellet. Wait 30 s before removing ethanol.
Repeat step 4 for a total of 2 washes.
Centrifuge briefly before returning to magnet and removing any residual ethanol.
Air dry for 2 min (DO NOT exceed 2 minutes as this will decrease elution efficiency).
Remove from the magnet and elute with 30.5 µL Buffer EB. Pipette mix 15X.
Incubate for 2 min at Room temperature.
Place the tube strip on the magnet until the solution clears.
Transfer30 µL to a new strip tube.
SECTION: 3&#x27; Gene Expression Library Construction
32. Sample Index PCR
Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. 
Add 50 µL Amp Mix to 30 µL sample.
Add 20 µL of an individual Dual Index TT Set A to each tube and record assignment. 
Pipette mix 5X (pipette set to 90 µl)  and centrifuge briefly. 
Incubate in a thermal cycler to incorporate sample indices (run time: approximately 25-40 minutes).
 

6.  Store at 4 °C for up to 4320 min or proceed to the next step.

SECTION: 3&#x27; Gene Expression Library Construction
33. Post Sample Index PCR Double Sided Size Selection -- SPRIselect 
Add 60 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15X (pipette set to 150 µl). 
Incubate for 5 min at -20 Room temperature. 
Place on magnet until solution clears. DO NOT discard supernatant. 
Transfer 150 µL supernatant to a new tube strip tube. 
Add 20 µL SPRIselect reagent (0.8X) to each sample and pipette mix 15x (pipette set to 150 µl). 
Incubate for 5 min at -20 Room temperature.
Place on magnet and remove supernatant. DO NOT discard any beads. 
Add 200 µL freshly prepared 80% ethanol to the pellet. Wait 30 s before removing ethanol.
Repeat step 8 for a total of 2 washes.
Centrifuge briefly before returning to magnet and removing any residual ethanol.
Air dry for 2 min (DO NOT exceed 2 minutes as this will decrease elution efficiency).
Remove from the magnet and elute with 35.5 µL Buffer EB. Pipette mix 15X.
Incubate for 2 min at Room temperature.
Place the tube strip on the magnet until the solution clears.
Transfer35 µL to a new strip tube.
Store at 4 °C for up to 4320 min or at -20 °C for long-term storage.
 

SECTION: ATAC Library Sequencing
34. (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
SECTION: ATAC Library Sequencing
35. 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
SECTION: Gene Expression Library Sequencing
36. (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
SECTION: Gene Expression Library Sequencing
37. 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
SECTION: Cell Ranger Arc Mapping and Analysis Pipeline
38. 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.