TEA-seq
Elliott Swanson, Lucas Graybuck, Peter J Skene
10x Genomics
Trimodal
Triple
scATAC-seq
scRNA-seq
ADT
Allen Institute
Allen Institute for Immunology
AIFI
BioLegend
TotalSeq
Disclaimer
This protocol is provided by the Allen Institute and contributors "as is" and any express or implied warranties, including, but not limited to, the implied warranties of merchantability and fitness for a particular purpose are disclaimed. In no event shall the Allen Institute or contributors be liable for any direct, indirect, incidental, special, exemplary, or consequential damages (including, but not limited to, procurement of substitute goods or services; loss of use, data, or profits; or business interruption) however caused and on any theory of liability, whether in contract, strict liability, or tort (including negligence or otherwise) arising in any way out of the use of this protocol, even if advised of the possibility of such damage.
Abstract
TEA-seq is a method for Transcriptomic, Epitope, and Accessibility measurement from thousands of single cells on the 10x Genomics Multiome platform. It generates scRNA-seq, scATAC-seq, and cell surface epitope (BioLegend TotalSeq-A) sequencing libraries linked by 10x cell barcodes for coordinated analysis. TEA-seq has been optimized for use with peripheral blood mononuclear cells (PBMCs).
Before start
-
Pre-chill a swinging-bucket rotor centrifuge to
4°Cfor use throughout antibody staining protocol, with holders for 5 mL polystyrene round-bottom tubes. -
If performing bead cleanup steps (36 and onward), remove Dynabeads MyOne SILANE reagent from storage at least
0h 30m 0sbefore beginning the cleanup to allow them to equilibrate to room temperature.
Steps
Buffer preparation
Stain Buffer Dulbecco's phosphate-buffered saline (DPBS) supplemented with 2% w/v bovine serum albumin.
Wash Buffer Final composition of 20 mM Tris HCl (Tris(hydroxymethyl)aminomethane hydrochloride) pH 7.4, 150 mM NaCl, 3 mM MgCl2.
Perm Buffer Wash Buffer (above) with the addition of digitonin to a final concentration of 0.01% w/v. Stocks of 5% w/v digitonin in DMSO can be prepared ahead of time and aliquots can be stored at -20°C for future use. We recommend limiting the number of freeze-thaw cycles to five.
Tagmentation Buffer Wash Buffer (above) with the addition of Protector RNase Inhibitor at a final concentration of 1U / µl.
Sample preparation
Remove cryopreserved cells from liquid nitrogen storage and thaw in a 37°C water bath for 3-5 minutes until no ice is visible.
Slowly dilute cells in 10 mL of pre-warmed 37°C
Centrifuge the cells at 400x g,4°C and remove the supernatant.
Resuspend the cell pellet in 1 mL of ice cold Dulbecco's phosphate-buffered saline supplemented with 0.2% w/v bovine serum albumin (DPBS + 0.2% BSA) by pipette mixing. Bring the volume to 10 mL with DPBS + 0.2% BSA.
Centrifuge the cells at 400x g,4°C and remove the supernatant.
Resuspend the cell pellet in 1 mL of ice cold Dulbecco's phosphate-buffered saline supplemented with 0.2% w/v bovine serum albumin (DPBS + 0.2% BSA) by pipette mixing. Dilute the cell suspension in DPBS + 0.2% BSA as desired, targetting a cell concetration of 1 - 5 million cells / mL.
Count cells before proceeding to Antibody Staining.
Antibody staining
Centrifuge cells (recommended input of 1.5 - 5 million) at 400x g,4°C in a 5 mL polystyrene round bottom tube and remove the supernatant.
Resuspend the cell pellet in ice cold Stain Buffer (DPBS + 2% BSA) by gently pipette mixing.
Add 10 ul of TruStain FcX and pipette mix thoroughly. Incubate cells for 0h 10m 0s .
Centrifuge antibodies at 14000x g,4°C to remove antibody aggregates. Avoid pipetting from the bottom of the tube when aspirating antibodies following centrifugation.
Add TotalSeq-A antibodies and pipette mix thoroughly. Incubate cells for 0h 30m 0s.
Dilute cells with 4 mL of ice cold Stain Buffer and gently mix.
Centrifuge cells at 400x g,4°C and remove the supernatant.
Resuspend the cell pellet in 4 mL of ice cold Stain Buffer by gently pipette mixing.
Centrifuge cells at 400x g,4°C and remove the supernatant.
Repeat steps 16-17 for a total of three washes.
Resuspend the cell pellet in 1 mL of ice cold Stain Buffer and count the cells.
Permeabilization
Aliquot 1 million stained cells into a 1.5 mL low bind tube and centrifuge at 400x g,4°C . Remove the supernatant.
Resuspend the cell pellet in 100 µl of ice cold Perm Buffer by pipette mixing. Incubate cells for 0h 5m 0s.
Dilute cells with 1mL of ice cold Wash Buffer and gently mix.
Centrifuge cells at 400x g,4°C and remove the supernatant.
Resuspend the cell pellet in 50 - 100 µl of ice cold Tagmentation Buffer depending on the desired cell input into the GEM barcoding reaction. An input of 15k cells requires a final concentration of 3k cells per µl.
Filter cell suspension using a 35 µm cell strainer to remove cell clumps. Transfer filtered solution to a new tube.
Count the cells and calculate the volume of cell suspension required for the desired cell input.
Tagmentation and GEM generation
Aliquot the desired number of cells to a new 96-well skirted PCR plate and bring volume to 5 µl with Tagmentation Buffer.
Create a master mix containing 7 µl of ATAC Buffer B and 3 µl of ATAC Enzyme B. Add 10 µl of master mix to each sample and gently pipette mix ten times.
Seal the plate and incubate on a thermal cycler for 1h 0m 0s at 37°C . Hold the reaction at 4°C upon completion. Allow samples to cool to 4°C , then proceed immediately to GEM generation (step 33).
Perform steps 30 - 32 during the incubation time.
During the tagmentation reaction remove the Single Cell Multiome Gel Beads, Template Switch Oligo, Reducing Agent B, and Barcoding Reagent Mix from storage and allow them to thaw at Room temperature .
Remove the Barcoding Enzyme Mix from storage 10 - 15 minutes before the end of the tagmentation reaction and maintain On ice.
Create a barcoding master mix on ice as detailed in the 10x Multiome ATAC + Gene Expression user guide (step 2.1 of the guide).
Load a Chromium Next GEM Chip J into a secondary holder. Dispense 50% Glycerol into unused chip wells as follows:
Row 1: 70 µl
Row 2: 50 µl
Row 3: 40 µl
Remove the sample plate from the thermal cycler once it reaches 4°C and maintain On ice .
Add 60 µl of master mix to each sample well and gently pipette mix five times.
Load 70 µl of master mix plus sample into row 1 of the chip.
Vortex the Multiome Gel Beads for 0h 0m 30s and add 50 µl to row 2 of the chip.
Add 45 µl of Partitioning Oil to row 3 of the chip.
Place a gasket over the loaded chip and load it onto the Chromium Controller. Initiate the run.
At completion of the run, slowly aspirate 100 µl of GEMs from the chip and slowly dispense into a new half-skirt plate on ice as detailed in the 10x Multiome ATAC + Gene Expression user guide (step 2.4 of the guide).
Incubate GEMs on a thermal cycler for 0h 45m 0s at 37°C, followed by 0h 30m 0s at 25°C , ending with a 4°C hold.
During the barcoding reaction remove the Quenching Agent from storage and equilibrate to Room temperature.
Add 5 µl of Quenching Agent to each sample and mix.
GEM cleanup
Remove Dynabeads MyOne SILANE reagent from storage at least 30 minutes before beginning the cleanup and allow it to equilibrate to Room temperature .
Remove the Cleanup Buffer from-20°C storage.
Heat at 65°C and thoroughly vortex until no precipitates are visible (approx. 0h 20m 0s).
Perform the Post GEM Incubation Dynabead cleanup as detailed in the 10x Multiome ATAC + Gene Expression User Guide (step 3.1 of the guide).
Perform a 2.0x bead:sample SPRIselect cleanup.
Bind beads for 0h 10m 0s at Room temperature .
Elute DNA off the beads by resuspending in 45.5 µl of Buffer EB.
Transfer 45 µl of sample to a new plate.
Pre-Amplification
Remove Pre-Amp Primers from storage and allow them to thaw at room temperature.
Remove Pre-Amp Mix from storage and thaw on ice.
Prepare a 0.2 µM solution of ADT-Rev-AMP and/or Additive HTO Primer in Low TE Buffer.
When processing both HTOs and ADTs in parallel create an equal mixture of both the ADT-Rev-AMP and Additive HTO Primer. Each primer in the mixture should be at a concentration of 0.2 uM.
On ice prepare a Pre-Amp master mix consisting of:
50 µl of Pre-Amp Mix
4 µl of Pre-Amp Primers
1 µl of 0.2 µM ADT-Rev-AMP and/or Additive HTO Primer solution per sample
-20On ice add 55 µl of Pre-Amp master mix to 45 µl of sample and mix.
Incubate reactions on a thermal cycler using the following 7 cycle PCR protocol:
Lid Temperature: 105°C
Reaction Volume: 100 µl
| A | B | C |
|---|---|---|
| Step | Temperature | Time |
| 1 | 72°C | 5 min |
| 2 | 98°C | 3 min |
| 3 | 98°C | 20 sec |
| 4 | 63°C | 30 sec |
| 5 | 72°C | 1 min |
| 6 | - | Go to step 3 x6 (total of 7 cycles) |
| 7 | 72°C | 1 min |
| 8 | 4°C | Hold |
7 cycle Pre-Amp PCR protocol
Perform a 2.0x bead:sample SPRIselect cleanup.
Bind beads for 0h 10m 0s at Room temperature .
Elute DNA off the beads by resuspending in 160.5 µl of Buffer EB.
Transfer 160 µl of sample to a new plate.
ATAC Library Prep
Remove SI-PCR Primer B and Sample Index plate N, Set A from -20°C storage and allow them to thaw at room temperature.
Remove Amp Mix from-20°C storage and thaw on ice.
On ice , prepare a PCR master mix consisting of 50 µl Amp Mix and 7.5 µl SI-PCR Pimer B per sample.
On ice , add 57.5 µl of PCR master mix and 2.5 µl of an individual index to 40 µl of each pre-amplified sample. Mix thoroughly.
Incubate reactions on a thermal cycler using the following 9 cycle PCR protocol:
Lid Temperature: 105°C
Reaction Volume: 100 µl
| A | B | C |
|---|---|---|
| Step | Temperature | Time |
| 1 | 98°C | 45 sec |
| 2 | 98°C | 20 sec |
| 3 | 67°C | 30 sec |
| 4 | 72°C | 20 sec |
| 5 | - | Go to step 2 x8 (total of 9 cycles) |
| 6 | 72°C | 1 min |
| 7 | 4°C | Hold |
9 cycle ATAC indexing PCR protocol
Perform a dual-sided 0.6x/1.6x bead:sample SPRIselect size-selection clean-up as detailed in the 10x Multiome ATAC + Gene Expression User Guide (step 5.2 of the guide).
cDNA Amplification
Remove cDNA Primers from storage and allow them to thaw at room temperature.
Remove Amp Mix from storage and thaw On ice .
Prepare a 2 µM solution of ADT-Rev-AMP and/or Additive HTO Primer in Low TE Buffer.
When processing both HTOs and ADTs in parallel create an equal mixture of both the ADT-Rev-AMP and Additive HTO Primer. Each primer in the mixture should be at a concentration of 2 uM.
On ice , prepare a cDNA Amplification master mix consisting of:
50 µl of Amp Mix
15 µl of cDNA Primers
1 µl of 2 µM ADT-Rev-AMP and/or Additive HTO Primer solution per sample.
On ice , add 66 µl of cDNA Amplification master mix to 35 µl of each pre-amplified sample. Mix thoroughly.
Incubate reactions on a thermal cycler using the following 8 cycle PCR protocol:
Lid Temperature: 105°C
Reaction Volume: 101 µl
| A | B | C |
|---|---|---|
| Step | Temperature | Time |
| 1 | 98°C | 3 min |
| 2 | 98°C | 15 sec |
| 3 | 63°C | 20 sec |
| 4 | 72°C | 1 min |
| 5 | - | Go to step 2 x7 (total of 8 cycles) |
| 6 | 72°C | 1 min |
| 7 | 4°C | Hold |
8 cycle cDNA Amplification protocol
Perform a dual-sided 0.6x/2.0x bead:sample SPRIselect size-selection clean-up.
Add 60 µl of SPRIselect beads to each reaction, mix thoroughly, and incubate the plate at room temperature on the bench for 5 minutes .
Transfer the plate to a magnet and incubate on a magnet for 5 minutes or until the solution is clear.
Transfer the supernatant to a new half-skirt plate. The supernatant contains the ADT & HTO fragments and is retained for further processing.
Complete the SPRI cleanup on the large cDNA fragments by performing two washes with 80% ethanol.
Elute the cDNA off the beads by resuspending in 40.5 µl of Buffer EB.
Transfer 40 µl of each cDNA library to a new plate.
Add an additional 140 µl of SPRIselect beads to the ADT/HTO containing supernatant from the first cleanup. Carefully pipette mix ten times or until the solution is fully homogenous.
Incubate the plate at room temperature on the bench for 10 minutes .
Transfer the plate to a magnet and incubate on a magnet for 5 minutes or until the solution is clear.
Remove and discard the supernatant.
Perform two washes with 80% ethanol.
ADT or HTO only
Elute the ADTs or HTOs off the beads by resuspending in 45.5 µl of Buffer EB.
Transfer 45 µl of ADT solution to a new plate.
ADT & HTO
Elute the ADTs or HTOs off the beads by resuspending in 90.5 µl of Buffer EB.
Transfer 45 µl of the HTO/ADT solution to each of two new plates. HTO and ADT fragments will be amplified in separate indexing PCRs using 45 µl of starting material each.
Run 1 ul of each amplified cDNA library on an Agilent Bioanalyzer High Sensitivity chip at a 1:10 dilution.
Set a region at 200 - 9000 bp and record the concentration in pg / µl. Calculate the total cDNA yield in ng.
Consult the 10x Multiome ATAC + Gene Expression user guide (step 6.1) if adjusting the number of PCR cycles in the Gene Expression indexing reaction is necessary.
Proceed to step 64 for ADT indexing PCR or step 68 for HTO indexing PCR. When preparing both ADT and HTO libraries these sections can be performed in either order.
ADT indexing PCR
Remove KAPA HiFi HotStart ReadyMix from -20°C storage and thaw on ice.
Remove 10 µM SI-PCR Primer and 10 µM ADT-i7 primers from -20°C storage and thaw at room temperature.
On ice , add the following to each 45 µl ADT sample. Mix thoroughly.
50 µl KAPA HiFi HotStart ReadyMix
2.5 µl 10 µM SI-PCR Primer
2.5 µl of an individual 10 µM ADT-i7 primer
Incubate ADT reactions on a thermal cycler using the following 15 cycle PCR protocol:
Lid Temperature: 105°C
Reaction Volume: 100 µl
| A | B | C |
|---|---|---|
| Step | Temperature | Time |
| 1 | 95°C | 3 min |
| 2 | 95°C | 20 sec |
| 3 | 60°C | 30 sec |
| 4 | 72°C | 20 sec |
| 5 | - | Go to step 2 x14 (total of 15 cycles) |
| 6 | 72°C | 5 min |
| 7 | 4°C | Hold |
15 cycle ADT indexing PCR protocol
Perform a 1.6x bead:sample SPRIselect cleanup.
Bind beads for 5 minutes at room temperature.
Elute DNA off the beads by resuspending in 30.5 µl of Buffer EB.
Transfer 30 µl of final ADT library to a new plate.
HTO indexing PCR
Remove KAPA HiFi HotStart ReadyMix from -20°C storage and thaw on ice.
Remove 10 µM SI-PCR Primer and 10 µM HTO-i7 primers from -20°C storage and thaw at room temperature.
On ice , add the following to each 45 µl HTO sample. Mix thoroughly.
50 µl KAPA HiFi HotStart ReadyMix
2.5 µl 10 µM SI-PCR Primer
2.5 µl of an individual 10 µM HTO-i7 primer
Incubate reactions on a thermal cycler using the following 10 cycle PCR protocol:
Lid Temperature: 105°C
Reaction Volume: 100 µl
| A | B | C |
|---|---|---|
| Step | Temperature | Time |
| 1 | 95°C | 3 min |
| 2 | 95°C | 20 sec |
| 3 | 64°C | 30 sec |
| 4 | 72°C | 20 sec |
| 5 | - | Go to step 2 x9 (total of 10 cycles) |
| 6 | 72°C | 5 min |
| 7 | 4°C | Hold |
10 cycle HTO indexing PCR protocol
Perform a 1.6x bead:sample SPRIselect cleanup.
Bind beads for 5 minutes at room temperature.
Elute DNA off the beads by resuspending in 30.5 µl of Buffer EB.
Transfer 30 µl of final HTO library to a new plate.
Gene Expression Library Prep
Remove Fragmentation Buffer, Adapter Oligos, Ligation Buffer, and Dual Index Plate TT Set A from -20°C storage and thaw at room temperature.
Remove Fragmentation Enzyme, DNA Ligase, and Amp Mix immediately before use and maintain On ice.
On ice , prepare a fragmentation master mix conisting of 5 µl of Fragmentation Buffer and 10 µl of Fragmentation Enzyme per sample.
Transfer 10 µl of each amplified cDNA library to a new plate. On ice, add 25 µl of Buffer EB and 15 µl of fragmentation master mix to each sample. Mix thoroughly.
Incubate reactions on a pre-cooled thermal cycler holding at 4°C .
Skip to the next step to ramp to 32°C for 0h 5m 0s , followed by 65°C for 0h 30m 0s, ending with a 4°C hold.
Perform a dual-sided 0.6x/0.8x bead:sample SPRIselect size-selection clean-up as detailed in the 10x Multiome ATAC + Gene Expression User Guide (step 7.2 of the guide).
On ice , prepare an adapter ligation master mix consisting of
20 µl of Ligation Buffer
10 µl of DNA Ligase
20 µl of Adapter Oligos.
per sample.
On ice , add 50 µl of adapter ligation master mix to each sample well. Mix thoroughly.
Incubate reactions on a thermal cycler at 20°C for 0h 15m 0s, ending with a hold at 4°C.
Perform a 0.8x bead:sample SPRIselect cleanup as detailed in the 10x Multiome ATAC + Gene Expression User Guide (step 7.4 of the guide).
On ice , add 50 µl of Amp Mix and 20 µl of an individual Dual Index TT Set A to each sample.
Mix thoroughly.
Incubate reactions on a thermal cycler using the following 14 cycle PCR protocol:
Lid Temperature: 105°C
Reaction Volume: 100 µl
| A | B | C |
|---|---|---|
| Step | Temperature | Time |
| 1 | 98°C | 45 sec |
| 2 | 98°C | 20 sec |
| 3 | 54°C | 30 sec |
| 4 | 72°C | 20 sec |
| 5 | - | Go to step 2 x13 (total of 14 cycles) |
| 6 | 72°C | 1 min |
| 7 | 4°C | Hold |
14 cycle Gene Expression indexing PCR protocol
Perform a dual-sided 0.6x/0.8x bead:sample SPRIselect size-selection clean-up as detailed in the 10x Multiome ATAC + Gene Expression User Guide (step 7.6 of the guide).
Final Library QC
Run each ATAC, Gene Expression, and ADT final library on an Agilent Bioanalyzer High Sensitivity chip or equivalent electrophoresis assay.
Set regions around each library trace to estimate the average base-pair size of the library. Evaluate the traces for presence of primer-dimer of other processing artifacts.
Quantify library concentration via qPCR using the
Calculate a size-adjusted concentration (in pM) for each library using the electropherogram and qPCR data.
Library concentrations determined using methods other than qPCR such as PicoGreen may be less accurate for this assay, especially for ADT & HTO libraries.
Multiply the calculated average concentration with the following factor:
Size of DNA Standard in bp (452) / Average fragment length of library in bp
Sequencing
Sequence libraries on the desired Illumina instrument platform. ATAC libraries will require different i5 index read lengths depending on whether the platform uses the Forward Strand or Reverse Complement indexing workflow. The Reverse Complement workflow requires ATAC libraries to be sequenced on a separate flow cell with a longer i5 read length due to the spacer in the capture sequence.
Reference the 10x Genomics Q&A for instrument specific use of the Forward and Reverse indexing workflows.
Read length for Forward Strand indexing workflow joint flow cell (ATAC, Gene Expression, ADT)
Read 1: 50 bp
i7 Index: 10 bp
i5 Index: 16 bp
Read 2: 90 bp
Read length for Forward Strand indexing workflow ATAC only flow cell
Read 1: 50 bp
i7 Index: 8 bp
i5 Index: 16 bp
Read 2: 50 bp
Read length for Reverse Strand indexing workflow ATAC only flow cell
Read 1: 50 bp
i7 Index: 8 bp
i5 Index: 24 bp
Read 2: 50 bp
Read length for Gene Expression flow cell (with or without ADT or HTO)
Read 1: 28 bp
i7 Index: 10 bp
i5 Index: 10 bp
Read 2: 90 bp
Read length for ADT and/or HTO only flow cell
Read 1: 28 bp
i7 Index: 8 bp
Read 2: 15 bp
Sequencing Depth
We recommend a minimum read count of 30k for Gene Expression, 60k for ATAC, 12k for ADT, and 2k for HTO, per cell when sequencing PBMCs. Other cell types may require additional sequencing. In our experience the required sequencing depth of ADT libraries tends to scale with the size of the panel, with the exception of rare markers.
Reference the 10x Genomics Sequencing Requirements for Single Cell Multiome ATAC + Gene Expression support page for more details.
Demultiplexing
Demultiplexing of ATAC and Gene Expression data can be performed using either Cell Ranger mkfastq (10x Genomics) or bcl2fastq2 (Illumina). We prefer bcl2fastq2 as we have found that it significantly outperforms mkfastq in terms of processing time and flexibility. Flow cells containing a combination of ATAC, Gene Expression, ADT, or HTO libraries, must use bcl2fastq2 for demultiplexing.
Software
| Value | Label |
|---|---|
| bcl2fastq | NAME |
| Illumina | DEVELOPER |
| https://emea.support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html | LINK |
| 2.20 | VERSION |
Software
| Value | Label |
|---|---|
| Cell Ranger | NAME |
| 10x Genomics | DEVELOPER |
| https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest | LINK |
| 5.0 | VERSION |
The commands below may be used to demultiplex TEA-seq data by library type. Use of bcl2fastq2 requires a sample sheet in standard Illumina Experiment Manager format for each library type.
# demultiplex gene expression data
bcl2fastq --use-bases-mask=Y28n*,I10,I10n*,Y90n* \
--create-fastq-for-index-reads \
--minimum-trimmed-read-length=8 \
--mask-short-adapter-reads=8 \
--ignore-missing-positions \
--ignore-missing-filter \
--ignore-missing-bcls \
-r 24 -w 24 -p 80 \
-R ${FLOWCELL_DIR} \
--output-dir={OUTPUT_DIR} \
--interop-dir={INTEROP_DIR} \
--sample-sheet={GEX_SAMPLE_SHEET_PATH}
# demultiplex ATAC data
bcl2fastq --use-bases-mask=Y50n*,I8n*,n8Y16,Y50n* \
--create-fastq-for-index-reads \
--minimum-trimmed-read-length=8 \
--mask-short-adapter-reads=8 \
--ignore-missing-positions \
--ignore-missing-filter \
--ignore-missing-bcls \
-r 24 -w 24 -p 80 \
-R ${FLOWCELL_DIR} \
--output-dir={OUTPUT_DIR} \
--interop-dir={INTEROP_DIR} \
--sample-sheet={ATAC_SAMPLE_SHEET_PATH}
# demultiplex ADT and HTO data (can use the same sample sheet).
bcl2fastq --use-bases-mask=Y28n*,I8n*,n*,Y90n* \
--create-fastq-for-index-reads \
--minimum-trimmed-read-length=8 \
--mask-short-adapter-reads=8 \
--ignore-missing-positions \
--ignore-missing-controls \
--ignore-missing-filter \
--ignore-missing-bcls \
-r 24 -w 24 -p 80 \
-R {FLOWCELL_DIR} \
--output-dir={OUTPUT_DIR} \
--interop-dir={INTEROP_DIR} \
--sample-sheet={ADT_SAMPLE_SHEET_PATH}
These commands have been written into a Bash script for ease of use. It is required to have bcl2fastq2 added to the environment path before use.
Data Preprocessing and analysis
Please see our Github repository for downstream data processing and analysis code:
