TITLE: Single cell RNA sequencing (scRNA-seq)
AUTHORS: Edgar Otto, Celine C. Berthier, Paul Hoover

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><h2></h2></div><div class = "text-block">Previously, RNA sequencing for whole-genome gene expression analysis could only be performed on whole tissue (bulk RNA seq), or microdissected tissue compartments, where gene expression measurements reflect an average across all captured cell types. Single cell RNA sequencing allows the measurement and comparison of gene expression of individual cells and thus capture the previously underappreciated cellular heterogeneity within tissues such as the kidney. Our protocol allows the dissociation of a single kidney biopsy core to a single cell suspension. The total mRNA from each living cell is then captured, uniquely barcoded, and gene expression profiled with RNAseq. Single cell transcriptomics of adult kidney tissue is performed using 10X Genomics droplet-based technology. 10X Genomics technology combines microfluidics with molecular barcoding and custom bioinformatics software (CellRanger). High-throughput single cell transcriptomic measurements enable profiling of individual cell types. </div></div>

[STEPS]
?. [Freezing in Cryostor]
Remove biopsy frozen in  CryoStor solution in a  cryovial out of  the liquid nitrogen tank. Thaw frozen biopsy in a  water bath under constant swirling for 60-90 sec only until biopsy at the bottom is ice-free.
500 µl
1.8 ml
37 °C
?. [Thawing and Cell Dissociation]
Transfer biopsy to plastic petri dish (ø 3.5 cm) filled with 1 ml DMEM/F12/10%FBS  for  to wash off remaining DMSO at room temperature.
?. [Thawing and Cell Dissociation]
Transfer to second plastic petri dish (ø 3.5 cm) filled with 1 ml DMEM/F12/10%FBS and incubate for  at room temperature.
?. [Thawing and Cell Dissociation]
Take picture of biopsy together with a ruler.
?. [Thawing and Cell Dissociation]
Cut biopsy in ~1 mm long pieces using a scalpel.
?. [Thawing and Cell Dissociation]
Prepare dissociation medium:  DMEM/F12 (no FBS) and  µl Liberase TL (conc.: 2.5 mg/ml H2O, final enzyme conc.: 0.25 mg/ml in  Eppendorf tube (noBind).
450 µl
50 µl
1.5 ml
?. [Thawing and Cell Dissociation]
Transfer biopsy tissue to prepared dissociation medium prewarmed to .
37 °C
?. [Thawing and Cell Dissociation]
Incubate at  for  and shake at 500 rpm in an Eppendorf Thermomixer.
37 °C
?. [Thawing and Cell Dissociation]
Triturate after  for 15 times with wide bore  pipette tip.
1 ml
?. [Thawing and Cell Dissociation]
Stop reaction with DMEM/F12/10%FBS (room temperature) and gently mix.
500 µl
?. [Thawing and Cell Dissociation]
Incubate for  at room temperature.
?. [Thawing and Cell Dissociation]
Filter through 30 µm gaze (Miltenyi) into  tube on ice.
15 ml
?. [Thawing and Cell Dissociation]
Push tissue through gaze using a  syringe plunger (rubber side).
5 ml
?. [Thawing and Cell Dissociation]
Wash filter with cold DMEM/F12/10%FBS.
10 ml
?. [Thawing and Cell Dissociation]
Filter single cell suspension again through new 30 µm gaze (Miltenyi) into a second  tube.
15 ml
?. [Thawing and Cell Dissociation]
Wash first tube with  cold DMEM/F12/10% FBS and apply through 30 µm gaze into second  tube.
15 ml
1 ml
15 ml
?. [Thawing and Cell Dissociation]
Centrifuge cell suspension at 200 x g  for  at .
4 °C
?. [Thawing and Cell Dissociation]
Remove supernatant using glass Pasteur pipette and house vacuum. (~ remain)
20 µl
?. [Thawing and Cell Dissociation]
Add and dispense cell pellet carefully with  DMEM/F12/10%FBS.
40 µl
?. [Thawing and Cell Dissociation]
Take  for cell count and mix with Trypan blue.
5 µl
5 µl
?. [Thawing and Cell Dissociation]
Count viable cells on hemocytometer or with automated cell counter (Countess)
?. [Thawing and Cell Dissociation]
Submit to 10X Genomics Chromium platform.
?. [Thawing and Cell Dissociation]
Load up to 10,000 viable cells in up to  of the cell suspension to 10X Chromium array ASAP.
46 µl
?. [Freezing in Cryostor]
Place the  cryovial with  HypoThermosol on ice
1.8 ml
1.5 ml
?. [Freezing in Cryostor]
Add the renal biopsy specimen to the cryovial with HypoThermosol immediately.
?. [Freezing in Cryostor]
Keep upright and at  (on ice) between the collection step and the cryopreservation step.
4 °C
?. [Freezing in Cryostor]
Place the  cryovial with  CryoStor on ice.
1.8 ml
1.5 ml
?. [Freezing in Cryostor]
Transfer the renal biopsy specimen to the cryovial with CryoStor CS10 very quickly.
?. [Freezing in Cryostor]
Keep the cryovial upright and on ice for  to allow permeabilization.
?. [Freezing in Cryostor]
Place the cryovial in a Mr. Frosty freezing unit containing isopropanol.
?. [Freezing in Cryostor]
Place the Mr. Frosty container in the  freezer for .
-80 °C
?. [Freezing in Cryostor]
Transfer the cryovial from the Mr. Frosty container to liquid N2, submerged. (Cryovials can be shipped on dry ice)
?. [Thawing and Cell Dissociation]
Place the  cryovial with  HypoThermosol on ice
1.8 ml
1.5 ml
?. [Thawing and Cell Dissociation]
Add the renal biopsy specimen to the cryovial with HypoThermosol immediately.
?. [Thawing and Cell Dissociation]
Keep upright and at  (on ice) between the collection step and the cryopreservation step.
4 °C
?. [Thawing and Cell Dissociation]
Place the  cryovial with  CryoStor on ice.
1.8 ml
1.5 ml
?. [Thawing and Cell Dissociation]
Transfer the renal biopsy specimen to the cryovial with CryoStor CS10 very quickly.
?. [Thawing and Cell Dissociation]
Keep the cryovial upright and on ice for  to allow permeabilization.
?. [Thawing and Cell Dissociation]
Place the cryovial in a Mr. Frosty freezing unit containing isopropanol.
?. [Thawing and Cell Dissociation]
Place the Mr. Frosty container in the -80°C freezer for .
?. [Thawing and Cell Dissociation]
Transfer the cryovial from the Mr. Frosty container to liquid N2, submerged. (Cryovials can be shipped on dry ice)
?. [Cell Handling Recommendations]
A fully dissociated, single-cell suspension is essential for the analysis of single-cell transcriptomes. a)	Suspension cell lines, bead-enriched and flow-sorted cells can be used directly after washing. b)	Adherent cell lines require trypsin treatment first. c)	Single-cell suspension from tissues requires optimization of dissociation.
?. [Cell Handling Recommendations]
It is important to treat cells gently to minimize cell lysis and loss: a)	When cells lyse, the mRNA will contaminate other GEMs. b)	Wash and resuspend in PBS + 0.04% non-acetylated BSA to minimize cell loss during handling. c)	Wash cells 2x using a wide-bore pipette tip to remove ambient RNA and contaminants.
?. [Cell Handling Recommendations]
Use a cell strainer to remove clumps or debris from washed cells. a)	Cell debris and fibers can be counted as either dead or live cells, causing inaccurate counts. b)	Cell debris and large clumps may clog or wet the chip.
?. [Cell Handling Recommendations]
Quantitate cells accurately before loading the system. a)	Approximately 65% loaded cells will be recovered. b)	Recommended range: 500 to 10,000 recovered cells. c)	Under- or over-loading may impact application performance.
?. [Cell Handling Recommendations]
Store cells suspension on ice until you are ready to load the chip. a)	Prepare cell suspension right before you are going to use it. b)	Load cells as soon as possible after preparation, ideally within 30 min
?. [Cell Handling Recommendations]
A fully dissociated, single cell suspension is essential for the analysis of single cell transcriptomes. a)	Suspension cell lines, bead-enriched and flow-sorted cells can be used directly after washing. b)	Adherent cell lines require trypsin treatment first. c)	Single cell suspension from tissues requires optimization of dissociation.
?. [Cell Handling Recommendations]
It is important to treat cells gently to minimize cell lysis and loss: a)	When cells lyse, the mRNA will contaminate other GEMs. b)	Wash and re-suspend in PBS + 0.04% non-acetylated BSA to minimize cell loss during handling. c)	Wash cells 2x using a wide-bore pipette tip to remove ambient RNA and contaminants.
?. [Cell Handling Recommendations]
Use a cell strainer to remove clumps or debris from washed cells. a)	Cell debris and fibers can be counted as either dead or live cells, causing inaccurate counts. b)	Cell debris and large clumps may clog or wet the chip.
?. [Cell Handling Recommendations]
Quantitate cells accurately before loading the system. a)	Approximately 65% loaded cells will be recovered. b)	Recommended range: 500 to 10,000 recovered cells. c)	Under- or over-loading may impact application performance.
?. [Cell Handling Recommendations]
Store cells suspension on ice until you are ready to load the chip. a)	Prepare cell suspension right before you are going to use it. b)	Load cells as soon as possible after preparation, ideally within
?. [Washing Isolated Cells]
Transfer cells in media to a  Eppendorf tube
2 ml
?. [Washing Isolated Cells]
Spin down cells to form pellet •	Depending on cell size and concentration, pellet size varies
?. [Washing Isolated Cells]
Remove supernatant
?. [Washing Isolated Cells]
Gently add 1x PBS + 0.04% BSA away from cell pellet
?. [Washing Isolated Cells]
Gently pipette mix with Wide Bore pipette tip
?. [Washing Isolated Cells]
Repeat the wash one more time
?. [Washing Isolated Cells]
Spin down cells to form pellet
?. [Washing Isolated Cells]
Remove supernatant
?. [Washing Isolated Cells]
Resuspend cells in 1x PBS + 0.04% BSA with gentle pipette mix •	For accurate cell counting, do not invert tubes
?. [Washing Isolated Cells]
Adjust to desired cell concentration as indicated in section 3.
?. [Preparing Single Cell Master Mix]
Equilibrate Single Cell 3’ Gel Beads to room temperature for at least , thaw reagents
?. [Preparing Single Cell Master Mix]
Resuspend the RT primer in l low TE buffer and prepare the thermal cycler.
40 µl
?. [Preparing Single Cell Master Mix]
Prepare Master Mix on ice. Do NOT add cells yet.
?. [Loading the Single Cell 3' Chip]
Place the Chip in the Holder before loading reagents
?. [Loading the Single Cell 3' Chip]
Handle the chip by its edges, taking care to avoid contacting the bottom surface (this helps to mitigate frictional charging)
?. [Loading the Single Cell 3' Chip]
Align the beveled corners at upper left and insert chip under the guide at the left edge of the recess in the holder
?. [Loading the Single Cell 3' Chip]
Press down on the right side of the chip until the spring loaded clip engages
?. [Loading the Single Cell 3' Chip]
Close the Holder and lay flat on benchtop
?. [Loading Single Cell Master Mix]
Dispense the  of Master Mix into each well of an 8-tube strip on a chilled metal block resting on ice. Then add the calculated volume of water into each strip tube.
66.2 µl
?. [Loading Single Cell Master Mix]
The next step is critical to recovering the maximum number of cells. It is important to ensure that:  a)	all of the cells in the sample are suspended b)	the sample is homogenous  when adding the cell suspension volume to the Master Mix.
?. [Loading Single Cell Master Mix]
Estimate the volume of the cell suspension and set the pipette at half that volume for pipette mixing. Wide bore pipette tips are not necessary for this step
?. [Loading Single Cell Master Mix]
Pipette the required volume of cell suspension from the center of the suspension volume.
?. [Loading Single Cell Master Mix]
Add the appropriate volume (µl) of single cell suspension to each well of the tube strip containing the Master Mix.
?. [Loading Single Cell Master Mix]
With a pipette set to  gently pipette mix the combined cells and Master Mix 5 times while keeping the tube strip on a chilled metal block resting on ice.
90 µl
?. [Loading Single Cell Master Mix]
Without discarding the pipette tips, transfer  Master Mix containing cells to the wells in the row labeled 1, taking care not to introduce bubbles.
90 µl
?. [Chip Priming]
After loading Master Mix, wait for
?. [Chip Priming]
This wait time is required for correct priming of channels inside the Single Cell 3’ Chip
?. [Chip Priming]
Then, immediately proceed to loading the Gel Beads
?. [Preparing Single Cell 3' Gel Beads]
Snap the Single Cell 3’ Gel Bead Strip into a 10x Vortex Adapter and vortex for . a)	A 30 sec wait while vortexing the Single Cell 3’ Gel Bead Strip is required to ensure proper priming of the Master Mix containing cells in the Single Cell 3’ Chip.
?. [Preparing Single Cell 3' Gel Beads]
Remove the Single Cell 3’ Gel Bead Strip and flick in a sharp, downward motion to ensure maximum recovery. Confirm that there are no bubbles at the bottom of the tube and that liquid levels are uniform.
?. [Loading Gel Beads and Partitioning Oil]
Pipette l Gel Beads from Gel Bead Strip
40 µl
?. [Loading Gel Beads and Partitioning Oil]
Dispense Gel Beads in row labeled 2 a)	Dispense into bottom of well b)	Slowly pipette Gel Beads c)	Avoid air bubbles
?. [Loading Gel Beads and Partitioning Oil]
Transfer Partitioning Oil to a reservoir and load row labeled 3 a)	Once oil is added, run the chip within .  The less time the chip sits, the better. b)	NEVER forget to add full volume of oil – doing so can damage the Chromium™ Controller! c)	Don’t leave oil in reagent reservoir for too long – avoid evaporation and contamination by particulates
?. [Loading Gel Beads and Partitioning Oil]
Attach the 10x™ Gasket.  The notched cut should be at the top left corner. a)	Ensure the gasket holes are aligned with the chip wells. b)	Do not touch or “smooth” the top surface of the gasket. c)	Keep the assembly horizontal to avoid wetting the 10x Gasket with Partitioning Oil.
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Load assembled Chip, Holder and Gasket.
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Keep the Chip Holder horizontal during transfer to the instrument to avoid wetting the gasket with reagents.
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Confirm Chromium Single Cell A program and press play button to start run.
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
The Controller will chime at the completion of the run (~).
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Maintain an Eppendorf twin.tec® 96-Well PCR plate for GEM transfer on a chilled metal block resting on ice.
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Remove the Single Cell 3’ Chip from the Chromium Controller.
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Open the 10x Chip Holder and fold the lid back until it clicks to expose the wells at a 45-degree angle.
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Check for volume uniformity in the:  a)	Sample (1) b)	Gel Bead (2) and c)	Partitioning Oil (3)  wells remaining in the Single Cell 3’ Chip.
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Remove Gasket
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Fold and lock Chip Holder at 45° angle
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Transfer  GEMs to twinc.tec® PCR plate a)	Aspirate GEMs •	Within  of completed run •	Pipette tip just above lowest point in recovery well •	Do not take more than 100 µl or double-dip b)	Dispense into PCR Plate •	Pipette tip against side of well •	Dispense slowly (20 sec) to avoid air bubbles •	Keep plate on chilled block
100 µl
?. [Running the Chromium™ Controller and transferring GEMs from the Chip]
Withdraw pipette tips from the wells and verify that there is no air in the tips. GEMs should appear opaque and uniform across all channels.
?. [GEM-RT Incubation]
Seal plate with Bio-Rad PX1TM Plate Sealer a)	Red stripe facing up b)	A tight seal is critical to GEM stability
?. [GEM-RT Incubation]
Cycle in Bio-Rad C1000 TouchTM thermal cycler with Deep Well Module
?. [GEM-RT Incubation]
Store in the PCR plate at  for up to  before proceeding to Post GEM-RT Cleanup.
4 °C
?. [Post GEM-RT Clean up]
Add  of Recovery Agent directly to samples in PCR plate.
125 µl
?. [Post GEM-RT Clean up]
Wait .
?. [Post GEM-RT Clean up]
With the same tips, move entire volume to 8 tube strips. (If PCR strip tubes have been used for GEM-RT reaction, no volume transfer into a new 8 tube strip is necessary) a)	Make sure to extract everything from the 96 well plate b)	Do not vortex or pipette mix after adding Recovery Agent
?. [Post GEM-RT Clean up]
Slowly remove  Recovery Agent/Partitioning Oil (pink) from the bottom of the tubes and discard a)	Some Recovery Agent may remain b)	Be careful to not aspirate any aqueous phase
125 µl
?. [cDNA Amplification Reaction]
Set up cDNA Amplification Master Mix a)	The optimal number of cycles for the cDNA amplification reaction is a trade-off between generating sufficient mass and minimizing PCR amplification artifacts
?. [cDNA Amplification Reaction]
SPRI Cleanup
?. [cDNA Amplification Reaction]
Bioanalyzer QC. Optimal performance has been characterized on input cDNA with a mean length > 2 kb.
?. [cDNA Amplification Reaction]
Fragmentation, End Repair & A-tailing
?. [cDNA Amplification Reaction]
Double-Sided Size Selection – SPRIselect
?. [cDNA Amplification Reaction]
Adaptor Ligation
?. [cDNA Amplification Reaction]
Post Ligation Cleanup – SPRIselect
?. [cDNA Amplification Reaction]
Sample Index PCR
?. [cDNA Amplification Reaction]
Double-Sided Size selection – SPRIselect
?. [cDNA Amplification Reaction]
Post Library Construction QC
?. [Library Preparation]
Fragmentation, End Repair & A-tailing
?. [Library Preparation]
Double Sided Size Selection – SPRIselect
?. [Library Preparation]
Adaptor Ligation
?. [Library Preparation]
Post Ligation Cleanup – SPRIselect
?. [Library Preparation]
Sample Index PCR
?. [Library Preparation]
Double Sided Size selection – SPRIselect
?. [Library Preparation]
Post Library Construction QC
?. [scRNA-seq sequencing and data processing]
The library mRNA prep is sequenced on an Illumina HiSeq 4000 platform. The output from the sequencer is then analyzed using the 10XGenomics software CellRanger. This step includes the alignment of the reads to human genome (GRCH38). The raw file output from CellRanger is further processed using Seurat R package. The downstream processing includes: Log transformation, filtering out cells with  5000 genes, filtering out cells with mitochondrial content > 20%, scaling based on UMI count, mitochondrial read content, principal component analysis, unsupervised clustering based on significant pca components, tSNEplot visualization and differential expression analysis using Wilcox test. The library mRNA prep is sequenced on an Illumina HiSeq 4000 platform. The output from the sequencer is then analyzed using the 10XGenomics software CellRanger. This step includes the alignment of the reads to human genome (GRCH38). The raw file output from CellRanger is further processed using Seurat R package. The downstream processing includes: Log transformation, filtering out cells with  5000 genes, filtering out cells with mitochondrial content > 20%, scaling based on UMI count, mitochondrial read content, principal component analysis, unsupervised clustering based on significant pca components, tSNEplot visualization and differential expression analysis using Wilcox test.