May 29, 2018

Public workspaceCell Hashing

DOI
dx.doi.org/10.17504/protocols.io.nfzdbp6
  • Marlon Stoeckius1,
  • Peter Smibert1
  • 1New York Genome Center Technology Innovation Lab
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DOI: dx.doi.org/10.17504/protocols.io.nfzdbp6
External link: https://cite-seq.com/cell-hashing/
Protocol Citation: Marlon Stoeckius, Peter Smibert 2018. Cell Hashing. protocols.io https://dx.doi.org/10.17504/protocols.io.nfzdbp6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 25, 2018
Last Modified: May 29, 2018
Protocol Integer ID: 10457
Abstract
This protocol is for performing Cell Hashing only. 
Sample multiplexing and super-loading on single cell RNA-sequencing platforms.
Cell Hashing 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.


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Guidelines
For experiments involving cell hashing, we recommend using the cost per cell calculator from the Satija lab to plan experiments, determine number of hashes, number of cells to load, expected doublet rates (detected and undetected) and cost considerations. 
The protocol workflow is as follows: 
  1. Cell staining for Drop-seq or 10x Genomics
  2. Drop-seq (Macosko et al. , 2015) or 10x Genomics single cell 3’ v2 assay
  3. cDNA amplification
  4. Separating HTO-derived cDNAs (<180bp) and mRNA-derived cDNAs (>300bp)
  5. Amplifying HTO sequencing library
  6. Purification of PCR product
Sequencing Cell Hashing libraries:
We estimate that an average of 100 molecules of HTO per cell is sufficient to achieve useful information. The number of reads required to obtain 100 molecules depends on the complexity of the sequencing library (e.g. duplication rate). HTO and cDNA sequencing libraries can be pooled at desired proportions. To obtain sufficient read coverage for both libraries we typically sequence HTO libraries in 5-10% of a lane and cDNA library fraction at 90% of a lane (HiSeq2500 Rapid Run Mode Flowcell).


Oligonucleotide sequences:
Hashtag oligos (HTOs):
These contain standard TruSeq DNA read 2 sequences and can be amplified using truncated versions of Illumina’s TruSeq DNA primer sets (see example D701_s below). See example below with a 12nt barcode:


Oligos required for HTO library amplification:
  • Drop-seq P5-SMART-PCR hybrid primer (for Drop-seq only)

  • 10x Genomics SI-PCR primer (for 10x Single Cell Version 2 only) 

  • HTO cDNA PCR additive primer     

  • Illumina TruSeq D701_s primer (for HTO amplification; i7 index 1, shorter than the original Illumina sequence)                                                                                                             

Materials
MATERIALS
ReagentFC blocking reagent (FcX)BioLegend
ReagentDesalting columns BioRad SciencesCatalog #732-6221
Reagent8-strip PCR tubes, e​mulsion safe (!)​USA ScientificCatalog #1402-4700
ReagentBioanalyzer chips and reagents (DNA High Sensitivity and small RNA kit)Agilent TechnologiesCatalog #5067-1548
ReagentSPRIselect reagentGe HealthcareCatalog #B23317
Reagent E-gel 4% Invitrogen - Thermo Fisher
ReagentLow-bind 1.5 mL tubes
ReagentPCR Thermocycler BioRad SciencesCatalog #T100
ReagentMagnetic tube rackInvitrogen - Thermo Fisher
ReagentQubit Invitrogen - Thermo Fisher
ReagentHemocytometer (e.g. Fuchs Rosenthal)
ReagentDMSO
ReagentPBS
ReagentTween20
ReagentBiotin
ReagentTE pH 8.0
ReagentBSA
Reagent80% Ethanol
Safety warnings
Please refer to the SDS (Safety Data Sheet) for hazard information.
Before start
Prepare Staining buffer (2%BSA/0.02%Tween, PBS). 
Cell staining for Drop-seq or 10x Genomics
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. 
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!
Resuspend ~1-2 million cells in 100 µl Staining buffer (2%BSA/0.02%Tween, PBS).
Amount100 µL Staining buffer
Add 10 µl Fc Blocking reagent (FcX, BioLegend).
Amount10 µL Fc Blocking reagent
Incubate for 10 minutes at 4˚C.
Temperature4 °C Incubation
Duration00:10:00 Incubation
Add 1 µg of single cell hashing antibody to cells. 
Amount1 µg Single cell hashing antibody
Incubate for 30 minutes at 4˚C.
Temperature4 °C Incubation
Duration00:30:00 Incubation
Wash cells with 1 mL Staining buffer (2%BSA/0.02%Tween, PBS). (1/3) 
Amount1 mL Staining buffer
Spin 5 minutes 400g at 4˚C. (1/3) 
Temperature4 °C Spinning
Duration00:05:00 Spinning
Wash cells with 1 mL Staining buffer. (2/3) 
Amount1 mL Staining buffer
Spin 5 minutes 400g at 4˚C. (2/3) 
Temperature4 °C Spinning
Duration00:05:00 Spinning
Wash cells with 1 mL Staining buffer. (3/3) 
Amount1 mL Staining buffer
Spin 5 minutes 400g at 4˚C. (3/3) 
Temperature4 °C Spinning
Duration00:05:00 Spinning
Resuspend cells in PBS at appropriate concentration for downstream application.
Note
​E.g. ​for 10x​ ~500 cells/µl; for Drop-seq​ [~200 cells/µl]; for super-loading​ ~1,500 cells/µl or higher.
Filter cells through 40 µm strainers (e.g. Flowmi cell strainer).
Verify cell concentration by counting on hemocytometer after filtration.
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
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 (<180bp) and mRNA-derived cDNAs (>300bp)
​Separation HTO-derived cDNAs (<180bp) and mRNA-derived cDNAs (>300bp)
Perform SPRI selection to separate mRNA-derived and antibody-oligo-derived cDNAs.
DO NOT DISCARD SUPERNATANT FROM 0.6X SPRI. THIS CONTAINS THE HASHTAGS.
Add 0.6X SPRI to cDNA reaction as described in 10x Genomics or Drop-seq protocol.
Incubate 5 minutes and place on magnet.
Duration00:05:00 Incubation on magnet
Supernatant contains hashtags.
Beads contain full length mRNA-derived cDNAs.
mRNA-derived cDNA >300bp (beads fraction)
mRNA-derived cDNA >300bp (beads fraction)
Proceed with standard 10x or Drop-seq protocol for cDNA sequencing library preparation.
For hashtags <180bp (supernatant fraction), follow the sections below.
Purify Hashtags using two 2X SPRI purifications
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.
Transfer entire volume into a low-bind 1.5 mL tube.
Incubate 10 minutes at room temperature.
Duration00:10:00 Incubation
Place tube on magnet and wait ~2 minutes until solution is clear. 
Duration00:02:00 Magnet
Carefully remove and discard the supernatant.
Add 400 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (only one Ethanol wash).
Amount400 µL 80% Ethanol
Duration00:00:30 Ethanol wash
Carefully remove and discard the ethanol wash.
Centrifuge tube briefly and return it to magnet.
Remove and discard any remaining ethanol.
Resuspend in beads in 50 µl water. 
Amount50 µL Water
Perform another round of 2X SPRI purification by adding 100 µl SPRI reagent directly onto resuspended beads.
Amount100 µL SPRI reagent
Mix by pipetting. 
Incubate 10 minutes at room temperature. 
Duration00:10:00 Incubation
Place tube on magnet and wait ~2 minutes until solution is clear.
Duration00:02:00 Magnet
Carefully remove and discard the supernatant.
Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (1st Ethanol wash).
Amount200 µL 80% Ethanol
Duration00:00:30 1. Ethanol wash
Carefully remove and discard the ethanol wash.
Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (2nd Ethanol wash).
Amount200 µL 80% Ethanol
Duration00:00:30 2. Ethanol wash
Carefully remove and discard the ethanol wash.
Centrifuge tube briefly and return it to magnet. 
Remove and discard any remaining ethanol. 
Allow the beads to air dry for 2 minutes (do not over dry beads).
Duration00:02:00 Air drying
Resuspend beads in 45 µl water. 
Amount45 µL Water
Pipette mix vigorously and incubate at room temperature for 5 minutes.
Duration00:05:00 Incubation
Place tube on magnet and transfer clear supernatant into two PCR tubes.
Amplify HTO sequencing library
Amplify HTO sequencing library
Prepare 100 µL PCR reaction with purified small fraction as follows: 
First, add 45 µl purified Hashtag fraction. 
ReagentAmount
purified Hashtag fraction45 µl
2x KAPA Hifi PCR Master Mix55 µl
TruSeq DNA D7xx_s primer (containing i7 index) 10 µM2.5 µl
P5 oligo at 10 µM depending on application*2.5 µl
* For Drop-seq use P5-SMART-PCR hybrid oligo.  For 10x use SI PCR oligo.
Amount45 µL Purified Hashtag fraction
Add 50 µl 2x KAPA Hifi PCR Master Mix.
Amount50 µL 2x KAPA Hifi PCR Master Mix
Add 2.5 µl TruSeq DNA D7xx_s primer (containing i7 index) 10 µM.
Amount2.5 µL TruSeq DNA D7xx_s primer (containing i7 index) 10 µM
Add 2.5 µl P5 oligo at 10 µM depending on application:
▪ For Drop-seq use P5-SMART-PCR hybrid oligo.
▪ For 10x use SI PCR oligo.
Cycling conditions:
95˚C   3 min 95˚C   20 sec 64˚C   30 sec 72˚C   20 sec 72˚C   5 min   | |         ~ 8-12 cycles | 
Purification
Purification
Purify PCR product using 1.6X SPRI purification by adding 160 µl SPRI reagent.
Amount160 µL SPRI reagent
Incubate 5 minutes at room temperature.
Duration00:05:00 Incubation
Place tube on magnet and wait 1 minute until solution is clear.
Duration00:01:00 Magnet
Carefully remove and discard the supernatant.
Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (1st Ethanol wash).
Amount200 µL 80% Ethanol
Duration00:00:30 1. Ethanol wash
Carefully remove and discard the ethanol wash.
Add 200 µl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (2nd Ethanol wash).
Amount200 µL 80% Ethanol
Duration00:00:30 2. Ethanol Wash
Carefully remove and discard the ethanol wash.
Centrifuge tube briefly and return it to magnet.
Remove and discard any remaining ethanol. 
Allow the beads to air dry for 2 minutes.
Duration00:02:00 Air drying
Resuspend beads in 20 µl water.
Amount20 µL Water
Pipette mix vigorously and incubate at room temperature for 5 minutes.
Duration00:05:00 Incubation
Place tube on magnet and transfer clear supernatant to PCR tube.
Hashtag libraries are now ready to be sequenced.
Quantify library by standard methods (QuBit, BioAnalyzer, qPCR).
Expected result
Hashtag library will be around 180 bp (Figure 1).