May 29, 2018
CITE-seq and Cell Hashing
- Marlon Stoeckius1,
- Peter Smibert1
- 1New York Genome Center Technology Innovation Lab
- Human Cell Atlas Method Development Community
External link: https://cite-seq.com/protocol/
Protocol Citation: Marlon Stoeckius, Peter Smibert 2018. CITE-seq and Cell Hashing. protocols.io https://dx.doi.org/10.17504/protocols.io.nhqdb5w
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 27, 2018
Last Modified: May 29, 2018
Protocol Integer ID: 10512
Abstract
This protocol is for performing CITE-seq and Cell Hashing in parallel.
CITE-seq:
Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) is a multimodal single cell phenotyping method developed in the Technology Innovation lab at the New York Genome Center in collaboration with the Satija lab.
CITE-seq uses DNA-barcoded antibodies to convert detection of proteins into a quantitative, sequenceable readout. Antibody-bound oligos act as synthetic transcripts that are captured during most large-scale oligodT-based scRNA-seq library preparation protocols (e.g. 10x Genomics, Drop-seq, ddSeq).
This allows for immunophenotyping of cells with a potentially limitless number of markers and unbiased transcriptome analysis using existing single-cell sequencing approaches.
Cell Hashing:
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.
Attachments
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.
Sequencing CITE-seq and Hashing libraries:
We estimate that an average of 100 molecules per ADT or HTO per cell is sufficient to achieve useful information, we typically sequence our ADT / HTO libraries to obtain significantly more reads than this per cell. The number of reads required to obtain 100 molecules depends on the complexity of the sequencing library (e.g. duplication rate). ADT, HTO and cDNA sequencing libraries can be pooled at desired proportions. We typically sequence ADT at 10% and HTO libraries at 5% of a lane and cDNA library fraction at 85% of a lane (HiSeq2500 Rapid Run Mode Flowcell).
Oligonucleotide sequences:
CITE-seq antibody-oligos (ADTs):
CITE-seq antibody-oligos contain standard small TruSeq RNA read 2 sequences and can be amplified using Illumina’s Truseq Small RNA primer sets (RPIx – primers, see example RPI1 below). See example below with a 12nt barcode:
Hashtag barcoding antibody-oligos (HTOs):
Cell Hashing antibody-oligos 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 ADT and 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)
- ADT cDNA PCR additive primer
- HTO cDNA PCR additive primer
- Illumina Small RNA RPI1 primer (for ADT amplification; i7 index 1, Oligonucleotide sequences © 2015 Illumina, Inc)
- Illumina TruSeq D701_s primer (for HTO amplification; i7 index 1, shorter than the original Illumina sequence)
Materials
MATERIALS
FC blocking reagent (FcX)BioLegend
8-strip PCR tubes, emulsion safe (!) (e.g. TempAssure PCR 8-strips)USA Scientific
Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies
SPRIselect reagent GE HealthcareCatalog #B23317
E-gel 4% Invitrogen - Thermo Fisher
Low-bind 1.5 mL tubes
PCR Thermocycler (e.g. T100)Bio-Rad Laboratories
Magnetic tube rack Invitrogen - Thermo Fisher
Qubit Invitrogen - Thermo Fisher
Hemocytometer (e.g. Fuchs Rosenthal)
DMSO
PBS
Tween20
TE pH 8.0
BSA (DNAse, RNAse and protease free) VWR International (Avantor)Catalog #0332-25G
Dead Cell Removal Kit Miltenyi Biotec
80% 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. Dead Cell Removal kit) is recommended.
Resuspend ~1-2 million cells in 100 µl Staining buffer (2%BSA/0.02%Tween, PBS).
100 µL Staining buffer
Add 10 µl Fc Blocking reagent (FcX, BioLegend).
10 µL Fc Blocking reagent
Incubate for 10 minutes at 4˚C.
4 °C Incubation
00:10:00 Incubation
While cells are incubating in Fc Block, prepare antibody-pool using ~0.5 - 1 µg (or titrated amounts) of each CITE-seq antibody and 1 µg of single cell hashing antibody (pool).
Add antibody-oligo pool to cells.
Incubate for 30 minutes at 4˚C.
4 °C Incubation
00:30:00 Incubation
Wash cells with 1 mL Staining buffer. (wash 1/3)
1 mL Staining buffer
Spin 5 minutes 400g at 4˚C. (wash 1/3)
4 °C Spinning
00:05:00 Spinning
Wash cells with 1 mL Staining buffer. (wash 2/3)
1 mL Staining buffer
Spin 5 minutes 400g at 4˚C. (wash 2/3)
4 °C Spinning
00:05:00 Spinning
Wash cells with 1 mL Staining buffer. (wash 3/3)
1 mL Staining buffer
Spin 5 minutes 400g at 4˚C. (wash 3/3)
4 °C Spinning
00:05:00 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
cDNA amplification
Add “additive” primers to cDNA PCR to increase yield of ADT and/or HTO products:
ADT PCR additive primer (2 µM): 1 µl (for 10x Genomics) or 0.4 µl (for Drop-seq)
HTO PCR additive primer (1 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.
Separating ADT / HTO-derived cDNAs (<180bp) and mRNA-derived cDNAs (>300bp)
Separating ADT / 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 ADTs and hashtags!
Add 0.6X SPRI to cDNA reaction as described in 10x Genomics or Drop-seq protocol.
Incubate 5 minutes and place on magnet.
00:05:00 Incubation on magnet
● Supernatant contains ADTs and 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 ADTs and Hashtags <180bp (supernatant fraction), follow the sections below.
Purifying ADTs using two 2X SPRI purifications
Purifying ADTs using two 2X SPRI purifications
To purify ADTs 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.
00:10:00 Incubation
Place tube on magnet and wait ~2 minutes until solution is clear
00:02:00 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).
400 µL 80% Ethanol
00:00:30 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.
50 µL Water
Perform another round of 2X SPRI purification by adding 100 µl SPRI reagent directly onto resuspended beads.
100 µL SPRI reagent
Mix by pipetting.
Incubate 10 minutes at room temperature.
00:10:00 Incubation
Place tube on magnet and wait ~2 minutes until solution is clear.
00:02:00 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).
200 µL 80% Ethanol
00:00:30 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).
200 µL 80% Ethanol
00:00:30 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).
00:02:00 Air drying
Resuspend beads in 90 µl water.
90 µL Water
Pipette mix vigorously.
Incubate mix at room temperature for 5 minutes.
00:05:00 Incubation
Place tube on magnet and transfer clear supernatant into two PCR tubes.
Amplifying ADT sequencing library
Amplifying ADT sequencing library
Prepare 100 µl PCR reaction with purified ADTs as follows:
First, add 45 µl purified ADT/Hashtag fraction.
| Reagent | Amount | |
| purified ADT/Hashtag fraction | 45 µl | |
| 2x KAPA Hifi PCR Master Mix | 50 µl | |
| TruSeq Small RNA RPIx primer (containing i7 index) 10 µM | 2.5 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.
45 µL Purified ADT/Hashtag fraction
Add 50 µl 2x KAPA Hifi PCR Master Mix.
50 µL 2x KAPA Hifi PCR Master Mix
Add 2.5 µl TruSeq Small RNA RPIx primer (containing i7 index) 10 µM.
2.5 µL TruSeq Small RNA RPIx primer (containing i7 index) 10 µM
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 60˚C 30 sec 72˚C 20 sec 72˚C 5 min | | | ~ 6-10 cycles | |
Amplifying HTO sequencing library
Amplifying HTO sequencing library
To prepare 100 µl of PCR reaction with purified small fraction, first, add 45 µl purified ADT/Hashtag fraction.
| Reagent | Amount | |
| purified ADT/Hashtag fraction | 45 µl | |
| 2x KAPA Hifi PCR Master Mix | 50 µl | |
| TruSeq DNA D7xx_s primer (containing i7 index) 10 µM | 2.5 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.
45 µL Purified ADT/Hashtag fraction
Add 50 µl 2x KAPA Hifi PCR Master Mix.
50 µL 2x KAPA Hifi PCR Master Mix
Add 2.5 µl TruSeq DNA D7xx_s primer (containing i7 index) 10 µM.
2.5 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 | |
Purifying PCR product
Purifying PCR product
Purify PCR product using 1.6X SPRI purification by adding 160 µl SPRI reagent.
160 µL SPRI reagent
Incubate 5 minutes at room temperature.
00:05:00 Incubation
Place tube on magnet and wait 1 minute until solution is clear.
00:01:00 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).
200 µL 80% Ethanol
00:00:30 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).
200 µL 80% Ethanol
00:00:30 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.
00:02:00 Air drying
Resuspend beads in 20 µl water.
20 µL Water
Pipette mix vigorously.
Incubate mix at room temperature for 5 minutes.
00:05:00 Incubation
Place tube on magnet and transfer clear supernatant to PCR tube.
ADT and Hashtag libraries are now ready to be sequenced.
Quantify libraries by standard methods (QuBit, BioAnalyzer, qPCR).
Expected result
ADT and Hashtag libraries will be around 180 bp (Figure 1 and 2).