TITLE: TEA-seq
AUTHORS: Elliott Swanson, Lucas Graybuck, Peter Skene

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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).</div></div>

[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  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.
-20 °C
Digitonin concentration was optimized for PBMCs. Permeabilization of different sample types may require further optimization. See Guidelines for suggestions.
?. [Sample preparation]
Remove cryopreserved cells from liquid nitrogen storage and thaw in a  water bath for 3-5 minutes until no ice is visible.
37 °C
This cell thaw protocol has been optimized for cryopreserved PBMCs. Alternate cell types or preservation methods may require different sample preparation procedures.
?. [Sample preparation]
Slowly dilute cells in 10 mL of pre-warmed  adding the first 3 mL dropwise.
37 °C
?. [Sample preparation]
Centrifuge the cells at  and remove the supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Sample preparation]
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.
?. [Sample preparation]
Centrifuge the cells at  and remove the supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Sample preparation]
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.
?. [Sample preparation]
Count cells before proceeding to Antibody Staining.
?. [Antibody staining]
Centrifuge cells (recommended input of 1.5 - 5 million) at  in a 5 mL polystyrene round bottom tube and remove the supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Antibody staining]
Resuspend the cell pellet in ice cold Stain Buffer (DPBS + 2% BSA) by gently pipette mixing.
?. [Antibody staining]
Add 10 ul of TruStain FcX and pipette mix thoroughly. Incubate cells for  .
[on ice]
?. [Antibody staining]
Add TotalSeq-A antibodies and pipette mix thoroughly. Incubate cells for .
[on ice]
?. [Antibody staining]
Dilute cells with 4 mL of ice cold Stain Buffer and gently mix.
?. [Antibody staining]
Centrifuge cells at   and remove the supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Antibody staining]
Resuspend the cell pellet in 4 mL of ice cold  Stain Buffer by gently pipette mixing.
?. [Antibody staining]
Centrifuge cells at  and remove the supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Antibody staining]
Repeat steps 16-17 for a total of three washes.
?. [Antibody staining]
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  . Remove the supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Permeabilization]
Resuspend the cell pellet in 100 µl of ice cold Perm Buffer by pipette mixing. Incubate cells for .
[on ice]
?. [Permeabilization]
Dilute cells with 1mL of ice cold Wash Buffer and gently mix.
?. [Permeabilization]
Centrifuge cells at   and remove the supernatant.
Centrifuge: 400 34, 5 min, 4 10
?. [Permeabilization]
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.
?. [Permeabilization]
Filter cell suspension using a 35 µm cell strainer to remove cell clumps. Transfer filtered solution to a new tube.
?. [Permeabilization]
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.
?. [Tagmentation and GEM generation]
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.
?. [Tagmentation and GEM generation]
Seal the plate and incubate on a thermal cycler for  at  . Hold the reaction at   upon completion. Allow samples to cool to  , then proceed immediately to GEM generation (step 33).Perform steps 30 - 32 during the incubation time.
37 °C
4 °C
4 °C
?. [Tagmentation and GEM generation]
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  .
0 Room temperature
First use of the Template Switch Oligo requires resuspension in 80 µl of Low TE Buffer.
?. [Tagmentation and GEM generation]
Remove the Barcoding Enzyme Mix from storage 10 - 15 minutes before the end of the tagmentation reaction and maintain .
on ice
?. [Tagmentation and GEM generation]
Create a barcoding master mix on ice as detailed in the 10x Multiome ATAC + Gene Expression user guide (step 2.1 of the guide).
?. [Tagmentation and GEM generation]
Load a Chromium Next GEM Chip J into a secondary holder. Dispense 50% Glycerol into unused chip wells as follows:Row 1: 70 µlRow 2: 50 µlRow 3: 40 µl
?. [Tagmentation and GEM generation]
Remove the sample plate from the thermal cycler once it reaches   and maintain  . Add 60 µl of master mix to each sample well and gently pipette mix five times.
4 °C
on ice
?. [Tagmentation and GEM generation]
Load 70 µl of master mix plus sample into row 1 of the chip.
?. [Tagmentation and GEM generation]
Vortex the Multiome Gel Beads for   and add 50 µl to row 2 of the chip.
?. [Tagmentation and GEM generation]
Add 45 µl of Partitioning Oil to row 3 of the chip.
?. [Tagmentation and GEM generation]
Place a gasket over the loaded chip and load it onto the Chromium Controller. Initiate the run.
?. [Tagmentation and GEM generation]
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).
?. [Tagmentation and GEM generation]
Incubate GEMs on a thermal cycler for  at , followed by   at   , ending with a  hold.
37 °C
25 °C
4 °C
?. [Tagmentation and GEM generation]
Add 5 µl of Quenching Agent to each sample and mix.
Optional Stopping Point: GEMs can be stored at   for for up to four weeks.
?. [GEM cleanup]
Remove Dynabeads MyOne SILANE reagent from storage at least 30 minutes before beginning the cleanup and allow it to equilibrate to  .
0 Room temperature
?. [GEM cleanup]
Remove the Cleanup Buffer from storage. Heat at   and thoroughly vortex until no precipitates are visible (approx. ).
-20 °C
65 °C
?. [GEM cleanup]
Perform the Post GEM Incubation Dynabead cleanup as detailed in the 10x Multiome ATAC + Gene Expression User Guide (step 3.1 of the guide).
?. [GEM cleanup]
Perform a 2.0x bead:sample SPRIselect cleanup. Bind beads for   at  . Elute DNA off the beads by resuspending in 45.5 µl of Buffer EB. Transfer 45 µl of sample to a new plate.
0 Room temperature
?. [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.
Primers can be prepared ahead of time and frozen at  .
?. [Pre-Amplification]
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
on ice
?. [Pre-Amplification]
add 55 µl of Pre-Amp master mix to 45 µl of sample and mix.
on ice
?. [Pre-Amplification]
Incubate reactions on a thermal cycler using the following 7 cycle PCR protocol:Lid Temperature: 105°CReaction Volume: 100 µl  ABC1StepTemperatureTime2172°C5 min3298°C3 min4398°C20 sec5463°C30 sec6572°C1 min76-Go to step 3 x6 (total of 7 cycles)8772°C1 min984°CHold
ABC1StepTemperatureTime2172°C5 min3298°C3 min4398°C20 sec5463°C30 sec6572°C1 min76-Go to step 3 x6 (total of 7 cycles)8772°C1 min984°CHold
Optional Stopping Point: Following amplification, reactions can be held at   for up to 72 hours.
?. [Pre-Amplification]
Perform a 2.0x bead:sample SPRIselect cleanup. Bind beads for   at  . Elute DNA off the beads by resuspending in 160.5 µl of Buffer EB. Transfer 160 µl of sample to a new plate.
0 Room temperature
Optional Stopping Point: Following cleanup, libraries can be held at   for up to 72 hours or stored at   .
?. [ATAC Library Prep]
Remove SI-PCR Primer B and Sample Index plate N, Set A from  storage and allow them to thaw at room temperature. Remove Amp Mix from storage and thaw on ice.
-20 °C
-20 °C
?. [ATAC Library Prep]
, prepare a PCR master mix consisting of 50 µl Amp Mix and 7.5 µl SI-PCR Pimer B per sample.
on ice
?. [ATAC Library Prep]
, 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.
on ice
?. [ATAC Library Prep]
Incubate reactions on a thermal cycler using the following 9 cycle PCR protocol:Lid Temperature: 105°CReaction Volume: 100 µl  ABC1StepTemperatureTime2198°C45 sec3298°C20 sec4367°C30 sec5472°C20 sec65-Go to step 2 x8 (total of 9 cycles)7672°C1 min874°CHold
ABC1StepTemperatureTime2198°C45 sec3298°C20 sec4367°C30 sec5472°C20 sec65-Go to step 2 x8 (total of 9 cycles)7672°C1 min874°CHold
Optional Stopping Point: Following amplification, reactions can be held at   for up to 72 hours.
?. [ATAC Library Prep]
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).
Optional Stopping Point: Final ATAC libraries can be stored at   .
?. [cDNA Amplification]
Remove cDNA Primers from storage and allow them to thaw at room temperature. Remove Amp Mix from storage and thaw  . 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
Primers can be prepared ahead of time and frozen at  .
?. [cDNA Amplification]
, prepare a cDNA Amplification master mix consisting of: 50 µl of Amp Mix15 µl of cDNA Primers1 µl of 2  µM ADT-Rev-AMP and/or Additive HTO Primer solution per sample.
on ice
?. [cDNA Amplification]
, add 66 µl of cDNA Amplification master mix to 35 µl of each pre-amplified sample. Mix thoroughly.
on ice
?. [cDNA Amplification]
Incubate reactions on a thermal cycler using the following 8 cycle PCR protocol:Lid Temperature: 105°CReaction Volume: 101 µl  ABC1StepTemperatureTime2198°C3 min3298°C15 sec4363°C20 sec5472°C1 min65-Go to step 2 x7 (total of 8 cycles)7672°C1 min874°CHold
ABC1StepTemperatureTime2198°C3 min3298°C15 sec4363°C20 sec5472°C1 min65-Go to step 2 x7 (total of 8 cycles)7672°C1 min874°CHold
Optional Stopping Point: Following amplification, reactions can be held at   for up to 72 hours.
?. [cDNA Amplification]
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 onlyElute 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 & HTOElute 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.
Optional Stopping Point: Following cleanup, amplified cDNA can be held at   for up to 72 hours or stored at   .
?. [cDNA Amplification]
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.
?. [ADT indexing PCR]
Remove KAPA HiFi HotStart ReadyMix from  storage and thaw on ice. Remove 10 µM SI-PCR Primer and 10 µM ADT-i7 primers from   storage and thaw at room temperature.
-20 °C
-20 °C
?. [HTO indexing PCR]
Incubate reactions on a thermal cycler using the following 10 cycle PCR protocol:Lid Temperature: 105°CReaction Volume: 100 µl  ABC1StepTemperatureTime2195°C3 min3295°C20 sec4364°C30 sec5472°C20 sec65-Go to step 2 x9 (total of 10 cycles)7672°C5 min874°CHold
ABC1StepTemperatureTime2195°C3 min3295°C20 sec4364°C30 sec5472°C20 sec65-Go to step 2 x9 (total of 10 cycles)7672°C5 min874°CHold
Optional Stopping Point: Following amplification, reactions can be held at   for up to 72 hours.
?. [HTO indexing PCR]
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.
Optional Stopping Point: Final HTO libraries can be stored at   .
?. [Gene Expression Library Prep]
Remove Fragmentation Buffer, Adapter Oligos, Ligation Buffer, and Dual Index Plate TT Set A from  storage and thaw at room temperature. Remove Fragmentation Enzyme, DNA Ligase, and Amp Mix immediately before use and maintain .
-20 °C
on ice
?. [Gene Expression Library Prep]
, prepare a fragmentation master mix conisting of 5 µl of Fragmentation Buffer and 10 µl of Fragmentation Enzyme per sample.
on ice
?. [Gene Expression Library Prep]
Transfer 10 µl of each amplified cDNA library to a new plate. , add 25 µl of Buffer EB and 15 µl of fragmentation master mix to each sample. Mix thoroughly.
on ice
?. [Gene Expression Library Prep]
Incubate reactions on a pre-cooled thermal cycler holding at  . Skip to the next step to ramp to   for  , followed by  for ,  ending with a  hold.
4 °C
32 °C
65 °C
4 °C
?. [Gene Expression Library Prep]
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).
?. [Gene Expression Library Prep]
, prepare an adapter ligation master mix consisting of 20 µl of Ligation Buffer10 µl of DNA Ligase20 µl of Adapter Oligos.per sample.
on ice
?. [Gene Expression Library Prep]
, add 50 µl of adapter ligation master mix to each sample well. Mix thoroughly.
on ice
?. [Gene Expression Library Prep]
Incubate reactions on a thermal cycler at  for , ending with a hold at .
20 °C
4 °C
?. [Gene Expression Library Prep]
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).
?. [Gene Expression Library Prep]
, add 50 µl of Amp Mix and 20 µl of an individual Dual Index TT Set A to each sample. Mix thoroughly.
on ice
?. [Gene Expression Library Prep]
Incubate reactions on a thermal cycler using the following 14 cycle PCR protocol:Lid Temperature: 105°CReaction Volume: 100 µl  ABC1StepTemperatureTime2198°C45 sec3298°C20 sec4354°C30 sec5472°C20 sec65-Go to step 2 x13 (total of 14 cycles)7672°C1 min874°CHold
ABC1StepTemperatureTime2198°C45 sec3298°C20 sec4354°C30 sec5472°C20 sec65-Go to step 2 x13 (total of 14 cycles)7672°C1 min874°CHold
If desired, the number of PCR cycles can be altered depending on cDNA yield. Reference the 10x Multiome ATAC + Gene Expression User Guide (step 7.5 of the guide) for vendor recommendations.
Optional Stopping Point: Following amplification, reactions can be held at   for up to 72 hours.
?. [Gene Expression Library Prep]
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).
Optional Stopping Point: Final Gene Expression libraries can be stored at   .
?. [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.
We recommend loading libraries at a 1:10 dilution for greater accuracy.
?. [Final Library QC]
Quantify library concentration via qPCR using the  or equivalent kit. Specifications will vary by qPCR instrument.
?. [Final Library QC]
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.https://kb.10xgenomics.com/hc/en-us/articles/360056364852-Should-I-select-Workflow-A-or-Workflow-B-for-the-i5-index-sequence-Read length for Forward Strand indexing workflow joint flow cell (ATAC, Gene Expression, ADT)Read 1: 50 bpi7 Index: 10 bpi5 Index: 16 bpRead 2: 90 bpRead length for Forward Strand indexing workflow ATAC only flow cellRead 1: 50 bpi7 Index: 8 bpi5 Index: 16 bpRead 2: 50 bpRead length for Reverse Strand indexing workflow ATAC only flow cellRead 1: 50 bpi7 Index: 8 bpi5 Index: 24 bpRead 2: 50 bpRead length for Gene Expression flow cell (with or without ADT or HTO)Read 1: 28 bpi7 Index: 10 bpi5 Index: 10 bpRead 2: 90 bpRead length for ADT and/or HTO only flow cellRead 1: 28 bpi7 Index: 8 bpRead 2: 15 bpSequencing DepthWe 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.https://support.10xgenomics.com/single-cell-multiome-atac-gex/sequencing/doc/specifications-sequencing-requirements-for-single-cell-multiome-atac-gene-expression
?. [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.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 databcl2fastq --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 databcl2fastq --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.
When demultiplexing adjust the read2 bases mask to be no longer than the read2 length (i.e. Y15n* for a 15 bp read 2). This applies to ADT and.or HTO only flow cells with a read2 length less than 90 bp.
ATAC demultiplexing requires a different --use-bases-mask for the i5 read depending on if the flow cell was sequenced using the Forward Strand or Reverse Complement indexing workflow. The bcl2fastq2 command above uses the base mask n8Y16 and assumes the flow cell was sequenced using the Reverse Complement workflow, consistent with the commonly used NovaSeq 6000 v1.5 platform. The Forward strand workflow will require the --use-bases-mask Y16 for the i5 read.
?. [Data Preprocessing and analysis]
Please see our Github repository for downstream data processing and analysis code:https://github.com/AllenInstitute/aifi-swanson-teaseq/
?. [ADT indexing PCR]
, add the following to each 45 µl ADT sample. Mix thoroughly.50 µl KAPA HiFi HotStart ReadyMix2.5 µl 10 µM SI-PCR Primer 2.5 µl of an individual 10 µM ADT-i7 primer
on ice
?. [ADT indexing PCR]
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.
Optional Stopping Point: Final ADT libraries can be stored at  .
?. [HTO indexing PCR]
, add the following to each 45 µl HTO sample. Mix thoroughly.50 µl KAPA HiFi HotStart ReadyMix2.5 µl 10 µM SI-PCR Primer 2.5 µl of an individual 10 µM HTO-i7 primer
on ice
?. [HTO indexing PCR]
Remove KAPA HiFi HotStart ReadyMix from  storage and thaw on ice. Remove 10 µM SI-PCR Primer and 10 µM HTO-i7 primers from   storage and thaw at room temperature.
-20 °C
-20 °C
?. [ADT indexing PCR]
Incubate ADT reactions on a thermal cycler using the following 15 cycle PCR protocol:Lid Temperature: 105°CReaction Volume: 100 µlStepTemperatureTime195°C3 min295°C20 sec360°C30 sec472°C20 sec5-Go to step 2 x14 (total of 15 cycles)672°C5 min74°CHold
StepTemperatureTime195°C3 min295°C20 sec360°C30 sec472°C20 sec5-Go to step 2 x14 (total of 15 cycles)672°C5 min74°CHold
Optional Stopping Point: Following amplification, reactions can be held at   for up to 72 hours.
?. [cDNA Amplification]
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.
?. [Tagmentation and GEM generation]
During the barcoding reaction remove the Quenching Agent from storage and equilibrate to .
0 Room temperature
?. [Antibody staining]
Centrifuge antibodies at  to remove antibody aggregates. Avoid pipetting from the bottom of the tube when aspirating antibodies following centrifugation.
Centrifuge: 14000 34, 10 min, 4 10