TITLE: 10X Genomics Single-Nucleus RNA-Sequencing for Transcriptomic Profiling of Adult Human Tissues
AUTHORS: Sarah Urata, Blue Lake, Dinh Diep, Masato Hoshi, Sanjay Jain, Kun Zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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]. 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. Here we present a modified version of the published 10X protocol [1] that we have adapted for the processing of adult human kidney nuclei.</div><div class = "text-block">References </div><div class = "text-block">1. Chromium Single Cell 3' Reagent Kits v3 User Guide (Rev A) CG000183, support.10xgenomics.com.  </div><div class = "text-block">2. Lake et al. (2016). Science, doi:10.1126/science.aaf1204.  </div><div class = "text-block">3. Lake et al. (2018). Nature Biotechnology, doi:10.1038/nbt.4038. </div><div class = "text-block">4. Lake et al. (2019). Nature Communications, doi:10.1038/s41467-019-10861-2. </div></div>

[STEPS]
?. [Isolate Nuclei]
Resuspend nuclei in  to  of PBS + 0.1% RNase Inhibitor (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, 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.
?. [GEM Generation and Barcoding]
Prepare Reagents for use Equilibrate gel beads to room temperature for . Thaw, vortex, and centrifuge RT Reagent, Template Switch Oligo, and Reducing Agent B. Verify no precipitate in Reducing Agent B. Centrifuge RT Enzyme C before adding to Master Mix.
?. [GEM Generation and Barcoding]
Prepare Master MixAdd  Master Mix to each tube of a PCR 8-tube strip on ice.
33.4 µl
?. [GEM Generation and Barcoding]
Load Chromium Single Cell B Chip 1.  Assemble Chromium Chip B 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.  Use Cell Suspension Volume Calculator Table to add the appropriate volume of nuclease-free water and corresponding volume of nuclei stock to make working nuclei suspension 6.	Nuclei Loading           a.	Minimum: load 800 nuclei → target recovery 500 nuclei           b.	Maximum: load 1600 nuclei → target recovery 10000 nuclei 7.	Add working Nuclei Suspension to the Master Mix in PCR tubes on ice for  total volume 8.	Load Master Mix and Nuclei Suspension into the bottom center of each well in Row 1 without introducing bubbles. 9.	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. 10.	Load  gel beads in Row 2. Only puncture the foil seal for gel bead tubes being used. Dispense slowly and without introducing bubbles. 11.	Load partitioning oil into each Row 3 by pipetting two aliquots of . 12.	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
280 µl
46.6 µl
80 µl
75 µl
40 µl
280 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 100 uL 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.
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-RT 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-RT Cleanup & cDNA Amplification - Prepare Reagents for use Thaw, vortex, and centrifuge a tube of Reducing Agent B and cDNA primers.Maintain Amp Mix on ice after vortex and centrifuge Thaw Cleanup Buffer at  for  with shaking at max rpm then cool to room temperature.
65 °C
?. [GEM Generation and Barcoding]
Post GEM RT-Cleanup --Dynabead 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 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. Vortex and centrifuge briefly. Place sample on magnetic separator and remove the supernatant. Wash twice with freshly prepared 80% ethanol.Elute with 35.5 uL Elution Solution I. Transfer 35 uL to a new strip tube.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. Vortex and centrifuge briefly. Place sample on magnetic separator and remove the supernatant. Wash twice with freshly prepared 80% ethanol.Elute with 35.5 uL Elution Solution I. Transfer 35 uL to a new strip tube.
125 µl
125 µl
200 µl
?. [GEM Generation and Barcoding]
cDNA Amplification Prepare cDNA Amplification Mix on ice. Vortex and centrifuge briefly. Add   cDNA Amplification Reaction Mix to   sample.Pipette mix 15x to mix and centrifuge briefly.Incubate in a thermocycler to amplify cDNA.Add  cDNA Amplification Reaction Mix to  sample.Pipette mix 15x to mix and centrifuge briefly.Incubate in a thermocycler to amplify cDNA.
65 µl
35 µl
Use cycle number optimization table for total number of cyclesWhen using nuclei, increase the optimized number of cycles by one cycleMaximum: load 1600 nuclei - perform 12 cycles
?. [GEM Generation and Barcoding]
cDNA Cleanup --SPRIselect Perform a 0.6X beads purification with SPRIselect reagent Wash twice with freshly prepared 80% ethanolElute with  Qiagen Buffer EB.Transfer to a new strip tube.Store at  for up to  or at  for up to 4 weeks. Or proceed to the next step.
40.5 µl
40 µl
4 °C
-20 °C
?. [GEM Generation and Barcoding]
cDNA QC & QuantificationRun TapeStation to obtain concentration and size Expected size range for amplified cDNA: 400 to 2500 bp cDNA total yield range: 80 to 1000 ng
QC Cutoff: Minimum cDNA total yield of 80 ng
?. [3' Gene Expression Library Construction ]
Prepare Reagents for use Thaw, vortex, and centrifuge Fragmentation Buffer, Adaptor Oligos, Ligation Buffer, SI Primer. Maintain on ice Fragmentation Enzyme, DNA Ligase, and Amp Mix.
?. [3' Gene Expression Library Construction ]
Fragmentation, End Repair & A-tailing1.  Prepare Fragmentation Mix on ice. Pipette mix and centrifuge briefly. 2.  Transfer  purified cDNA to a new strip tube 3.  Add  Buffer EB to each sample4.  Add  Fragmentation Mix to each sample5.  Pipette mix, centrifuge briefly, and transfer to pre-cooled block. “SKIP” hold step to initiate the protocol
10 µl
Note that 10 uL (25%) cDNA sample is sufficient for generating 3' Gene Expression Library.  The remaining 30 uL (75%) cDNA sample can be stored at 4 C for up to 72 hours or at -20 C for up to 4 weeks for generating additional 3' Gene Expression Libraries.
25 µl
15 µl
?. [3' Gene Expression Library Construction ]
Post Fragmentation, End Repair & A-tailing Double Sided Size Selection --SPRIselect Vortex to resuspend the SPRIselect reagent. Add  SPRIselect reagent (0.6X) to each sample and pipette mix 15x. Incubate  at room temperature.Centrifuge briefly Place sample on magnetic separator. DO NOT discard supernatant.  Transfer   supernatant to a new tube strip tube. Vortex to resuspend the SPRIselect reagent. Add   SPRIselect reagent (0.8X) to each sample and pipette mix 15x. Place sample on magnetic separator and remove supernatant. DO NOT discard any beads. Wash twice with freshly prepared 80% ethanolTransfer  supernatant to a new tube strip tube. Vortex to resuspend the SPRIselect reagent. Add  SPRIselect reagent (0.8X) to each sample and pipette mix 15x. Place sample on magnetic separator and remove supernatant. DO NOT discard any beads. Wash twice with freshly prepared 80% ethanolElute with  Qiagen Buffer EB. Transfer  to a new strip tube.
30 µl
75 µl
10 µl
50.5 µl
50 µl
?. [3' Gene Expression Library Construction ]
Adaptor LigationPrepare Adaptor Ligation Mix on ice. Pipette mix and centrifuge briefly.Add   Adaptor Ligation Mix to sample. Pipette mix 15x. Centrifuge briefly.Incubate in a thermocycler to ligate adaptor.Add  Adaptor Ligation Mix to sample. Pipette mix 15x. Centrifuge briefly.
50 µl
?. [3' Gene Expression Library Construction ]
Post Ligation Cleanup --SPRIselect Perform a 0.8X beads purification with SPRIselect reagentWash twice with freshly prepared 80% ethanolElute with  Qiagen Buffer EB.Transfer  to a new strip tube.
30.5 µl
30 µl
?. [3' Gene Expression Library Construction ]
Sample Index PCR1.  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  Sample Index PCR Mix to the sample 4.  Add  of individual Chromium i7 Sample Index (from SI-GA plate) to each well. Record well assignment. Mix and centrifuge briefly. 5.  Incubate in a thermocycler to incorporate sample indices. 6.  Store at  for up to . Or proceed to the next step.
60 µl
10 µl
Use cycle number recommendation table for total number of cycles; recommended number of cycles is based on cDNA input
4 °C
?. [3' Gene Expression Library Construction ]
Post Sample Index PCR Double Sided Size Selection --SPRIselect Vortex to resuspend the SPRIselect reagent. Add  SPRIselect reagent (0.6X) to each sample and pipette mix 15x. Incubate  at room temperature. Centrifuge briefly Place sample on magnetic separator. DO NOT discard supernatant. Transfer   supernatant to a new tube strip tube. Vortex to resuspend the SPRIselect reagent. Add   SPRIselect reagent (0.8X) to each sample and pipette mix 15x. Incubate   at room temperature.Centrifuge briefly Place sample on 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 (0.8X) to each sample and pipette mix 15x. Incubate  at room temperature.Centrifuge briefly Place sample on magnetic separator and remove supernatant. DO NOT discard any beads. Wash twice with freshly prepared 80% ethanol xi.	Elute with  Qiagen Buffer EB.Transfer  to a new strip tube. Store at  for up to  or at  for long-term storage.
60 µl
150 µl
20 µl
35.5 µl
35 µl
4 °C
-20 °C
QC Post Library Construction:Quantify Library (e.g. using Qubit dsDNA HS Assay)Estimate Library size range (e.g. using TapeStation or BioAnalyzer) - exprected size range of 300 to 800 bp, average: 475 bp
?. [Sequencing]
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: 8 cycles         c.  i5 Index: 0 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
?. [Sequencing]
NovaSeq Sequencing (target - 25,000-50,000 reads per nucleus)1.  Paired End, Single Indexing         a.  Read 1: 28 cycles         b.  i7 Index: 8 cycles        c.  i5 Index: 0 cycles         d.  Read 2: 91 cycles2.  Library Loading         a.  10X recommended Loading concentration: 300 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]
Generate pre-mRNA reference  b.	Generate Sample Sheet  c.	Generate fastq filesd. Run count for each sample
IMPORTANT: for nuclei experiments, we need to use the reference files with pre-mrna in order to count intronic reads in the UMI counts
Use sample sheet generator provided by 10X Genomics to generate a "SampleSheet.csv":Sample Sheet Generator
Use mkfastq command
?. [Isolate Nuclei]
Dilute nuclei stock to be in the 700-1200 nuclei per uL range in total volume using the dilution guide
50 µl
If nuclei concentrations fall below target range, then centrifugation can be performed to increase nuclei concentrations to be within rangeStart with nuclei stock in PBS with 0.1% BSASpin 900xg for 10 minutesResusupend in appropriate volume PBSUse 10 uL to count; 50 uL for loadingQC cutoff: minimum of 10,000 nuclei (for 200 nuclei per uL in 50 uL total volume)
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 .01% BSA in nuclei stock
?. [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