TITLE: 10X Genomics Single-Nucleus Assay of Transposase Accessible Chromatin-Sequencing for Epigenetic Profiling of Adult Human Tissues
AUTHORS: Blue Lake, Dinh Diep, Kun Zhang, Sarah Urata

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">10x Genomics Single Cell ATAC (v1) 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). Chromatin accessibility sequencing (ATAC-seq) provides insight into the upstream regulatory landscape associated with open and accessible chromatin regions.</div><div class = "text-block">References </div><div class = "text-block">1. Lake et al. (2016). Science, doi:10.1126/science.aaf1204. </div><div class = "text-block">2. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038.</div><div class = "text-block">3. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2.</div><div class = "text-block">4. Chromium Single Cell ATAC Reagent Kits User Guide v1 (Rev B) CG000168, support.10xgenomics.com. </div><div class = "text-block">5. Lake and Zhang. Nuclei Isolation Protocol Applicable to Adult Human Kidney Tissues, kpmp.org/researcher-resources.</div></div>

[STEPS]
?. [Isolate Nuclei]
Prepare nuclei according to the protocol "Isolation of single nuclei from solid tissues" steps 1-14. dx.doi.org/10.17504/protocols.io.ufketkw
?. [Isolate Nuclei]
Resuspend nuclei in  to  of 1X Diluted Nuclei Buffer (volume depends on target concentration)
100 µl
1 mL
?. [Isolate Nuclei]
Count nuclei (e.g., BioRad T20 Cell Counter)
?. [Isolate Nuclei]
Check nuclei integrity under fluorescent microscope using DAPI channel. Nuclei should appear distinct, with 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.
?. [Isolate Nuclei]
Dilute nuclei stock to be in the 1000-3200 nuclei per uL range in total volume using the 1X Diluted Nuclei Buffer.
5 µl
If nuclei concentrations fall below the target range, then centrifugation can be performed to increase nuclei concentrations to be within rangeStart with nuclei stock in PBS with 0.1% BSA. Add 5-10X volumes of 1X Diluted Nuclei Buffer (e.g. add 1 mL if nuclei stock is 100 uL)Spin 900xg for 10 minutes (use 500xg if total #nuclei > 100k)Resuspend in an appropriate volume of the 1X Diluted Nuclei Buffer.Use 2-5 uL to count; 5 uL for loadingQC cutoff: minimum of 10,000 nuclei.
Caution:  BSA is necessary to avoid clumping and prevent nuclei loss from sticking to the tube during the spin down.  DO NOT USE more than 0.01% BSA in nuclei stock
?. [GEM Generation and Barcoding]
Prepare Reagents for use Equilibrate gel beads to room temperature for . Thaw, vortex, and centrifuge Barcoding Reagent and Reducing Agent B. Verify no precipitate in Reducing Agent B. Centrifuge Barcoding Enzyme briefly before adding to Master Mix.
?. [GEM Generation and Barcoding]
Prepare Master MixPrepare Master Mix on ice (follow the recipe from the User Guide). Pipette mix 15X and centrifuge briefly. Add  Master Mix to each tube of transposed nuclei on ice.
65 µl
?. [GEM Generation and Barcoding]
Load Chromium Single Cell E Chip 1.  Assemble Chromium Chip E 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           a.	 into unused wells in Row 1           b.   into unused wells in Row 2           c.	 into unused wells in Row 3 5.	Load Master Mix and Transposed Nuclei Suspension into the bottom center of each well in Row 1 without introducing bubbles. 6.	Snap the Gel Bead strip into a 10X Vortex Adapter. Vortex . 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. 7.	Load  gel beads in Row 2. Only puncture the foil seal for gel bead tubes being used. Dispense slowly and without introducing bubbles. 8.	Load partitioning oil into each Row 3 by pipetting two aliquots of . 9.	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.
75 µl
40 µl
240 µl
75 µl
40 µl
240 µl
140 µl
?. [GEM Generation and Barcoding]
Run the Chromium ControllerPress the eject button on the controller to eject.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 (~), the controller will chime. Proceed immediately to the next step.
?. [GEM Generation and Barcoding]
Transfer GEMs Chill strip tubes 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  GEMs from the lowest points of the Recovery Wells without creating a seal between the pipette tips and the wells. 6.  Slowly dispense (~) GEMs into the tube strip on ice with the pipette tips against the sidewalls of the wells.
100 µl
GEMs should appear opaque and uniform across all channels.  Excess partitioning oil (clear) in the pipette tips indicates a potential clog.
?. [GEM Generation and Barcoding]
GEM IncubationStore at  for up to or at  for up to a week. Or proceed to the next step.
4 °C
-20 °C
?. [GEM Generation and Barcoding]
Post GEM Incubation Cleanup  - Prepare Reagents for use Thaw Cleanup Buffer at  for  with shaking at max rpm then cool to room temperature.
65 °C
?. [GEM Generation and Barcoding]
Post GEM Cleanup --Dynabeads Add Recovery Agent to each sample at room temperature. DO NOT MIX. Wait . Carefully aspirate  of pink oil phase from the bottom of the tube. DO NOT aspirate any aqueous sample. Prepare Dynabeads Cleanup Mix (follow the recipe from the User Guide)Vortex and centrifuge mixture and add  to each sample. Pipette 5X to mix.Incubate at room temperature for ; pipette mix again  into incubation. Prepare Elution Solution I (follow the recipe from the User Guide). Vortex and centrifuge briefly. Wash twice with  freshly prepared 80% ethanol.Elute with  Elution Solution I. Transfer  to a new strip tube.
125 µl
125 µl
200 µl
300 µl
40.5 µl
40 µl
?. [Library Construction]
Sample Index PCRThaw, vortex, and centrifuge a tube of SI-Primer B and a tube of Amp Mix and store on ice.Prepare Sample Index PCR Mix on ice (follow the recipe from the User Guide). Vortex and centrifuge briefly. Add   Sample Index PCR Mix to   sample.Add   individual Chromium i7 Sample Index N, Set A to each sample.Pipette mix 15X to mix and centrifuge briefly.Incubate in a thermocycler to amplify DNA.Prepare Sample Index PCR Mix on ice (follow the recipe from the User Guide). Vortex and centrifuge briefly. Add  Sample Index PCR Mix to  sample.Add  individual Chromium i7 Sample Index N, Set A to each sample.Pipette mix 15X to mix and centrifuge briefly.Incubate in a thermocycler to amplify DNA.
57.5 µl
40 µl
2.5 µl
Use cycle number optimization table for total number of cyclesMaximum: load 1,600 nuclei - perform 12 cycles
?. [GEM Generation and Barcoding]
Post GEM Cleanup --SPRIselect Incubate sample with beads for  at room temperatureWash twice with  freshly prepared 80% ethanolElute with  Qiagen Buffer EB. Incubate for Bind beads to magnet and transfer eluant to a new strip tube.Store at  for up to  or at  for up to 4 weeks. Or proceed to the next step.
200 µl
40.5 µl
40 µl
4 °C
-20 °C
?. [Sequencing]
MiSeq Sequencing - QC for estimation of library quality and number of nuclei captured, target 10,000 reads per nucleus1.  Paired End, Single Indexing         a.  Read 1: 50 cycles        b.  i7 Index: 8 cycles         c.  i5 Index: 16 cycles         d.  Read 2: 50 cycles 2.  Library Loading         a.  10X recommended Loading concentration: 11 pM         b.  Optional: 1% PhiX 3.  Output         a.  22-25 million reads
?. [Sequencing]
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: 16 cycles         d.  Read 2: 50 cycles2.  Library Loading         a.  10X recommended Loading concentration: 280 pM         b.  Optional: 1% PhiX3.  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 Mapping and Analysis Pipeline]
a.	Generate Sample Sheet  b.	Generate fastq filesc. Run count for each sample
Use sample sheet generator provided by 10X Genomics to generate a "SampleSheet.csv":Sample Sheet Generator
Use mkfastq command, e.g.: cellranger-atac mkfastq --id=sample1  --run=/IlluminaOutput/Run --samplesheet=Samplesheet.csv
Use count command, e.g.:cellranger-atac count --id=sample1 --reference=/opt/refdata-cellranger-arc-GRCh38-2020-A-2.0.0                         --fastqs=/IlluminaOutput/Run/fastq_path   --sample=mysample  --localcores=8   --localmem=64
?. [Transposition of Nuclei]
Prepare transposition mix.Thaw, vortex and centrifuge ATAC buffer and store on ice.Centrifuge briefly ATAC Enzyme and store on ice.Prepare a master mix of   ATAC buffer and  ATAC Enzyme for each sample. Mix thoroughly.Aliquot  of prepared master mix each tube of a PCR 8-tube strip on ice.Add   of nuclei suspension for each sample to the tube of master mix on ice.Pipette to mix gently with P-20 set at   6X. Do not centrifuge.
7 µl
3 µl
10 µl
5 µl
10 µl
?. [Transposition of Nuclei]
Isothermal incubation1. Incubate in a thermcycler at  for   with lid set at  .
37 °C
50 °C
?. [Library Construction]
Post Sample Index PCR Double Sided Size Selection - SPRIselect Vortex to resuspend the SPRIselect reagent. Add  SPRIselect reagent (0.4X) to each sample and pipette mix 15X. Incubate  at room temperature.Centrifuge briefly Place sample on the magnetic separator. DO NOT discard the supernatant.  Transfer   supernatant to a new tube strip tube. Vortex to resuspend the SPRIselect reagent. Add   SPRIselect reagent (1.14X) to each sample and pipette mix 15X. Place sample on the magnetic separator and remove supernatant. DO NOT discard any beads. Transfer  supernatant to a new tube strip tube. Vortex to resuspend the SPRIselect reagent. Add  SPRIselect reagent (1.14X) to each sample and pipette mix 15X. Wash twice with  freshly prepared 80% ethanolElute with  Qiagen Buffer EB. Bind beads to magnet and transfer  eluant to a new strip tube.
40 µl
130 µl
74 µl
200 µl
20 µl
20 µl
Post Library Construction QC:Quantify library (e.g. using Qubit dsDNA HS assay).Estimate library size range (e.g. using TapeStation or Bioanalyzer) - expected size range is 170-700bp and including nucleosome pattern of at least 3 different bands/peaks.