TITLE: Cell Hashing
AUTHORS: Brenton Paolella

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for performing Cell Hashing only. </div><div class = "text-block">Sample multiplexing and super-loading on single cell RNA-sequencing platforms.</div><div class = "text-block"><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Cell Hashing</span></a><span> uses a series of oligo-tagged antibodies against ubiquitously expressed surface proteins with different barcodes to uniquely label cells from distinct samples, which can be subsequently pooled in one scRNA-seq run. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its sample of origin, and robustly identify doublets originating from multiple samples.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/tx2fw26.png" /></div></div>

[STEPS]
?. [Cell staining for Drop-seq or 10x Genomics]
Obtain all single cell suspensions from different samples/conditions that will be multiplexed in the run. Keep samples in separate tubes until after cell hashing and shortly before loading cells into the single cell RNA-seq instrument. When aiming to super-load the same sample into one run, divide the sample up into equal proportions before staining with distinct cell hashing antibodies. Keep cell suspensions on ice (unless otherwise stated) at all times.
?. [Cell staining for Drop-seq or 10x Genomics]
Carefully count all cells to ensure accurate quantitation.● Make note of cell viability (>95%) and also include dead cells in the total cell count!● If you observe many dead cells, live cell enrichment (e.g. by FACS) is recommended!Record cell count in cell count table
?. [Cell staining for Drop-seq or 10x Genomics]
Resuspend all cells in 100 µl Staining buffer (2%BSA/0.02%Tween, PBS).We assume ~200K to 1 million cells per well, which is less than max of what can be stainedNote this much more hashing Ab than needed. Biolegends says it can stain 1-2 million cells
[Staining buffer]
?. [Cell staining for Drop-seq or 10x Genomics]
Add 10 µl Fc Blocking reagent (FcX, BioLegend).
[Fc Blocking reagent]
?. [Cell staining for Drop-seq or 10x Genomics]
Incubate for 10 minutes at 4˚C.
[Incubation]
[Incubation]
?. [Cell staining for Drop-seq or 10x Genomics]
Add 2 uL (1 µg) of single cell hashing antibody to each tube of cells.
[Single cell hashing antibody]
?. [Cell staining for Drop-seq or 10x Genomics]
Incubate for 30 minutes at 4˚C.
[Incubation]
[Incubation]
?. [Cell staining for Drop-seq or 10x Genomics]
Wash cells with 1 mL Staining buffer (2%BSA/0.02%Tween, PBS). (1/3)
[Staining buffer]
?. [Cell staining for Drop-seq or 10x Genomics]
Wash cells with 1 mL Staining buffer. (2/3)
[Staining buffer]
?. [Cell staining for Drop-seq or 10x Genomics]
Wash cells with 1 mL Staining buffer. (3/3)
[Staining buffer]
?. [Cell staining for Drop-seq or 10x Genomics]
Spin 5 minutes 400g at 4˚C. (1/3)
[Spinning]
[Spinning]
?. [Cell staining for Drop-seq or 10x Genomics]
Spin 5 minutes 400g at 4˚C. (2/3)
[Spinning]
[Spinning]
?. [Cell staining for Drop-seq or 10x Genomics]
Spin 5 minutes 400g at 4˚C. (3/3)
[Spinning]
[Spinning]
?. [Cell staining for Drop-seq or 10x Genomics]
Resuspend cells in Cell capture buffer at appropriate concentration for downstream application.We estimate resuspending in 200 uL if we have ~300,000 cells reatined after staining and washing
​E.g. ​for 10x​ ~500 cells/µl; for Drop-seq​ [~200 cells/µl]; for super-loading​ ~1,500 cells/µl or higher. Prior to experiment estimate min and max cells expected for resuspension
?. [Cell staining for Drop-seq or 10x Genomics]
Filter cells through 40 µm strainers (e.g. Flowmi cell strainer).
?. [Cell staining for Drop-seq or 10x Genomics]
Verify cell concentration by counting on Countess after filtration.Record counts in cell count table
?. [Cell staining for Drop-seq or 10x Genomics]
Pool all different samples/conditions at desired proportions and immediately proceed to next step.
?. Run ​Drop-seq (Macosko ​et al.​, 2015) or ​10x Genomics single cell 3’ v2 assay as described until before cDNA amplification.
?. [cDNA amplification step]
Add “additive” primer to cDNA PCR to increase yield of HTO products: HTO PCR additive primer (2 µM): 1 µl (for 10x Genomics) or 0.4 µl (for Drop-seq) Subtract the total volume of additive primer from the water added to the PCR reaction.
?. [​Separation HTO-derived cDNAs (300bp)]
Perform SPRI selection to separate mRNA-derived and antibody-oligo-derived cDNAs.
?. [​Separation HTO-derived cDNAs (300bp)]
DO NOT DISCARD SUPERNATANT FROM 0.6X SPRI. THIS CONTAINS THE HASHTAGS.
?. [​Separation HTO-derived cDNAs (300bp)]
Add 0.6X SPRI to cDNA reaction as described in 10x Genomics or Drop-seq protocol.
?. [​Separation HTO-derived cDNAs (300bp)]
Incubate 5 minutes and place on magnet.
[Incubation on magnet]
?. [​Separation HTO-derived cDNAs (300bp)]
Supernatant contains hashtags.Beads contain full length mRNA-derived cDNAs.
?. [mRNA-derived cDNA >300bp (beads fraction)]
Proceed with standard 10x or Drop-seq protocol for cDNA sequencing library preparation.For hashtags
?. [Purify Hashtags using two 2X SPRI purifications]
Purify Hashtags using two 2X SPRI purifications per manufacturer protocol. First, add 1.4X SPRI to supernatant to obtain a final SPRI volume of 2X SPRI.From Dani/KCO we expect 100 uL initial volume, but got 150 uL so added 300 uL beads
[SPRI beads]
?. [Purify Hashtags using two 2X SPRI purifications]
Transfer entire volume into a low-bind 1.5 mL tube.
?. [Purify Hashtags using two 2X SPRI purifications]
Incubate 10 minutes at room temperature.
[Incubation]
?. [Purify Hashtags using two 2X SPRI purifications]
Place tube on magnet and wait ~2 minutes until solution is clear.
[Magnet]
?. [Purify Hashtags using two 2X SPRI purifications]
Carefully remove and discard the supernatant.
?. [Purify Hashtags using two 2X SPRI purifications]
Add 450 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (only one Ethanol wash).This is the same total volume as beads + supernatant
[80% Ethanol]
[Ethanol wash]
?. [Purify Hashtags using two 2X SPRI purifications]
Carefully remove and discard the ethanol wash.
?. [Purify Hashtags using two 2X SPRI purifications]
Centrifuge tube briefly and return it to magnet.
?. [Purify Hashtags using two 2X SPRI purifications]
Resuspend in beads in 50 µl water. deviation form KAtie becuase the 1.5 mL tub is too large to elute in 15 uL
[Water]
?. [Purify Hashtags using two 2X SPRI purifications]
Perform another round of 2X SPRI purification by adding 100 µl SPRI reagent directly onto resuspended beads.deviation form Katie - she uses 30 uL beads w/15 uL eluted H2O
[SPRI reagent]
?. [Purify Hashtags using two 2X SPRI purifications]
Mix by pipetting.
?. [Purify Hashtags using two 2X SPRI purifications]
Incubate 10 minutes at room temperature.
[Incubation]
?. [Purify Hashtags using two 2X SPRI purifications]
Place tube on magnet and wait ~2 minutes until solution is clear.
[Magnet]
?. [Purify Hashtags using two 2X SPRI purifications]
Carefully remove and discard the supernatant.
?. [Purify Hashtags using two 2X SPRI purifications]
Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (1st Ethanol wash).
[80% Ethanol]
[1. Ethanol wash]
?. [Purify Hashtags using two 2X SPRI purifications]
Carefully remove and discard the ethanol wash.
?. [Purify Hashtags using two 2X SPRI purifications]
Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (2nd Ethanol wash).
[80% Ethanol]
[2. Ethanol wash]
?. [Purify Hashtags using two 2X SPRI purifications]
Carefully remove and discard the ethanol wash.
?. [Purify Hashtags using two 2X SPRI purifications]
Centrifuge tube briefly and return it to magnet.
?. [Purify Hashtags using two 2X SPRI purifications]
Remove and discard any remaining ethanol.
?. [Purify Hashtags using two 2X SPRI purifications]
Allow the beads to air dry for 2 minutes (do not over dry beads).
[Air drying]
?. [Purify Hashtags using two 2X SPRI purifications]
Resuspend beads in 15 µl water.
[Water]
?. [Purify Hashtags using two 2X SPRI purifications]
Pipette mix vigorously and incubate at room temperature for 5 minutes.
[Incubation]
?. [Purify Hashtags using two 2X SPRI purifications]
Place tube on magnet and transfer clear supernatant into PCR tubes. ~2-5 min
For PCR typically add ~5 ng DNA into each PCR reactionset up 3 different reactions with different cycles 12, 15, 18 cycles to not over amplify1 uL of elution, or 10 ng total input if concentratedtypical input range 2-30 ng/uL - but doesn't necessarily correlate to number of cycles
?. [Amplify HTO sequencing library]
Prepare 50 µL PCR reaction with purified small fraction as follows: add ~1 µl purified Hashtag fraction.   AB1ReagentAmount2purified Hashtag fraction~1 µl32x KAPA Hifi PCR Master Mix25 µl4TruSeq DNA D7xx_s primer (containing i7 index) 10 µM1.25 µl5SI PCR oligo* 10 µM1.25 µl6H2Oto 50 uL ~21.5 uL if 1uL hashing fraction used* For Drop-seq use P5-SMART-PCR hybrid oligo
[Hashtag fraction]
AB1ReagentAmount2purified Hashtag fraction~1 µl32x KAPA Hifi PCR Master Mix25 µl4TruSeq DNA D7xx_s primer (containing i7 index) 10 µM1.25 µl5SI PCR oligo* 10 µM1.25 µl6H2Oto 50 uL ~21.5 uL if 1uL hashing fraction used
?. [Amplify HTO sequencing library]
Cycling conditions:  AB195˚C&nbsp; &nbsp;3 min
95˚C&nbsp; &nbsp;20 sec
64˚C&nbsp; &nbsp;30 sec
72˚C&nbsp; &nbsp;20 sec
72˚C&nbsp; &nbsp;2-5 min&nbsp;
|
|&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; 10, 15, 18 cycles
|&nbsp;
AB195˚C&nbsp; &nbsp;3 min
95˚C&nbsp; &nbsp;20 sec
64˚C&nbsp; &nbsp;30 sec
72˚C&nbsp; &nbsp;20 sec
72˚C&nbsp; &nbsp;2-5 min&nbsp;
|
|&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; 10, 15, 18 cycles
|&nbsp;
?. [Purification ]
Purify PCR product using 2X SPRI purification by adding 100 µl SPRI reagent.can do a 0.8x SPRI to clean up if primer dimer persists after QC in a pinch, but better to reamplify with reserved Hashing template
[SPRI reagent]
?. [Purification ]
Incubate 5 minutes at room temperature.
[Incubation]
?. [Purification ]
Place tube on magnet and wait 1 minute until solution is clear.
[Magnet]
?. [Purification ]
Carefully remove and discard the supernatant.
?. [Purification ]
Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (1st Ethanol wash).
[80% Ethanol]
[1. Ethanol wash]
?. [Purification ]
Carefully remove and discard the ethanol wash.
?. [Purification ]
Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (2nd Ethanol wash).
[80% Ethanol]
[2. Ethanol Wash]
?. [Purification ]
Carefully remove and discard the ethanol wash.
?. [Purification ]
Centrifuge tube briefly and return it to magnet.
?. [Purification ]
Remove and discard any remaining ethanol.
?. [Purification ]
Allow the beads to air dry for 2 minutes.
[Air drying]
?. [Purification ]
Resuspend beads in 15 µl water.
[Water]
?. [Purification ]
Pipette mix vigorously and incubate at room temperature for 5 minutes.
[Incubation]
?. [Purification ]
Place tube on magnet and transfer clear supernatant to PCR tube.NOTE did 1 PCR tube at a time holding bottom of tube at top edge of magnet
?. [Purification ]
Hashtag libraries are now ready to be sequenced.Quantify library by standard methods (QuBit, BioAnalyzer, qPCR).
90 bp peak is primer if under amp'd, larger products than 186 are generated when over amplified
Hashtag library will be around 180 bp (Figure 1).
?. [Cell staining for Drop-seq or 10x Genomics]
While cells are incubating in Fc Block, prepare antibody-pool using 1 µg (or titrated amounts) of each TotalSeq™ antibody and 1 µg of single cell hashing antibody (pool).To maximize performance, centrifuge the antibody pool at 14,000xg at 2 – 8°C for 10 minutes before adding to the cells.Carefully pipette out the liquid, avoiding the bottom of the tube, and add the TotalSeq™ antibody cocktail to the cell suspension.
?. [Cell staining for Drop-seq or 10x Genomics]
Count pooled cell suspension. For 10x superloading ~1500 cells/uL is ideal (or 1.5e6 cells/mL). Record counts in cell count table
?. [Purify Hashtags using two 2X SPRI purifications]
Quantify using qubit or nano drop (less sensitive, but usally ok)
?. [Purify Hashtags using two 2X SPRI purifications]
Transfer to PCR strip tube