TITLE: DAb-seq: Single-Cell DNA and Antibody Sequencing
AUTHORS: Benjamin Demaree, Cyrille Delley, Harish N. Vasudevan, Cheryl A.C. Peretz, David Ruff, Catherine C. Smith, Adam R Abate

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Studies of acute myeloid leukemia rely on DNA sequencing and immunophenotyping by flow cytometry as primary tools for disease characterization. However, leukemia tumor heterogeneity complicates integration of DNA variants and immunophenotypes from separate measurements. Here we introduce DAb-seq, a novel technology for simultaneous capture of DNA genotype and cell surface phenotype from single cells at high throughput, enabling direct profiling of proteogenomic states in tens of thousands of cells. To demonstrate the approach, we analyze the disease of three patients with leukemia over multiple treatment timepoints and disease recurrences. We observe complex genotype-phenotype dynamics that illustrate the subtlety of the disease process and the degree of incongruity between blast cell genotype and phenotype in different clinical scenarios. Our results highlight the importance of combined single-cell DNA and protein measurements to fully characterize the heterogeneity of leukemia.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/42873d4a470fa94e903d34a732cf78eb92a5c469a7639700912c714a2598d1a8/cppdbg3hp.png" /></div></div>

[STEPS]
?. [Conjugation of antibodies to oligonucleotide barcodes]
Resuspend each monoclonal antibody to  in  PBS.
100 µg
100 µl
Antibody must be ordered in protein-free, azide-free buffer, preferably plain PBS. Resuspended from lyophilized stock with trace amounts of trehalose (e.g. from R&D Systems) is also acceptable. For a list of antibodies used in the DAb-seq publication, see Supplemental Table 2 attachment.
?. [Conjugation of antibodies to oligonucleotide barcodes]
Prepare  DBCO/PEG/NHS linker in DMSO. After adding DMSO, vortex at  for  to allow linker to dissolve completely.
0 Room temperature
For long-term storage, aliquot DBCO linker in   volumes and store at  .
?. [Conjugation of antibodies to oligonucleotide barcodes]
Dilute DBCO linker to in water.
?. [Conjugation of antibodies to oligonucleotide barcodes]
To each 100 uL antibody solution, add  of 2.5 mM DBCO linker. Pipet up and down to mix.
1.07 µl
?. [Conjugation of antibodies to oligonucleotide barcodes]
Incubate linker/antibody solution for  at .
-20 Room temperature
?. [Conjugation of antibodies to oligonucleotide barcodes]
Add  PBS to linker/antibody solution and transfer to 50 kDA Amicon filter.
400 µl
?. [Conjugation of antibodies to oligonucleotide barcodes]
Centrifuge . Discard flow-through.
Centrifuge: 14000 34, 10 min
?. [Conjugation of antibodies to oligonucleotide barcodes]
Add  PBS to filter and invert filter in new collection tube. Centrifuge  to elute.
180 µl
Centrifuge: 1500 34, 5 min
?. [Conjugation of antibodies to oligonucleotide barcodes]
Resuspend lyophilized oligonucleotide to  in PBS.
Oligos must have 5’ azide group (IDT code: /5AzideN/) and be HPLC-purified. For full oligo sequence, including barcodes, see Supplemental Table 2 attachment.
?. [Conjugation of antibodies to oligonucleotide barcodes]
Add  oligo to the approximately  of filtered antibody solution.
6.67 µl
200 µl
?. [Conjugation of antibodies to oligonucleotide barcodes]
Incubate at  .
4 °C
16-20 h is an acceptable incubation time.
?. [Conjugation of antibodies to oligonucleotide barcodes]
Add  PBS to antibody-oligo conjugate and transfer to 50 kDa Amicon filter.
300 µl
?. [Conjugation of antibodies to oligonucleotide barcodes]
Centrifuge . Discard flow-through.
Centrifuge: 14000 34, 10 min
?. [Conjugation of antibodies to oligonucleotide barcodes]
Repeat Step 13 two additional times, adding  PBS for each wash. This is a total of three washes.
500 µl
?. [Conjugation of antibodies to oligonucleotide barcodes]
Add  PBS to filter and invert filter in new collection tube. Centrifuge  to elute.
30 µl
Centrifuge: 1500 34, 5 min
?. [Conjugation of antibodies to oligonucleotide barcodes]
Collect ~50 uL eluant and store in Protein LoBind tube at .
4 °C
?. [Conjugation of antibodies to oligonucleotide barcodes]
Verify conjugation using a Bioanalyzer Protein 230 kit or equivalent gel electrophoretic assay. A representative Bioanalyzer Protein 230 trace is shown below. A peak representing the unconjugated antibody is visible at ~155 kDa. Larger peaks represent antibodies conjugated to one, two, or more oligos.
?. [Staining cells]
Collect cells from culture or thaw from frozen according to cell-specific protocols. Resuspend cells in 5-10 mL PBS-F (D-PBS + 5% FBS) and count. It is highly recommended to measure cell viability with trypan blue or other exclusion assay.
Cells should be >90% viable before staining to ensure minimal cell loss during the washing steps.
?. [Staining cells]
Spin down 2 million cells in a 15 mL DNA LoBind tube for  . Aspirate the supernatant and resuspend the cell pellet in  PBS-F.
Centrifuge: 400 34, 4 min
180 µl
It is helpful to grate the tube gently against the TC hood to loosen the cell pellet prior to the addition of buffer.
?. [Staining cells]
Add  BioLegend Human TruStain FcX blocking solution,  of a  dextran sulfate solution, and  of  salmon sperm DNA. Pipet gently to mix.
10 µl
4 µl
4 µl
?. [Staining cells]
Incubate .
on ice
?. [Staining cells]
Add  of each antibody-oligo conjugate and incubate  .
0.5 µg
on ice
?. [Staining cells]
Perform five washes to remove excess unbound antibody. For each wash, add  PBS-F to the tube and centrifuge .
5 mL
Centrifuge: 400 34, 4 min
?. [Staining cells]
After final wash, resuspend cells in Mission Bio Cell Buffer at a final concentration of 3M cells/mL.
?. [Cell encapsulation and barcoding on the Tapestri instrument]
Follow cell encapsulation and barcoding procedure as described in the attached Mission Bio document: "Tapestri Single-Cell DNA AML User Guide".
Follow sections "Encapsulate Cells" (page 18) through "UV Treatment and Targeted PCR Amplification" (page 31). The DAb-seq protocol for these sections are unchanged from the conventional DNA-only workflow.
?. [Cleanup barcoded DNA and antibody products]
Perform Steps 6.1 through 6.7 in the attached Mission Bio protocol. DO NOT discard the Ampure XP supernatant in Step 6.8, as this contains the short antibody tags. Instead, for each tube, transfer the supernatant to a new 1.5 mL DNA LoBind tube.
?. [Cleanup barcoded DNA and antibody products]
Finish DNA library cleanup as described in the Mission Bio protocol. Elute in 30 uL water and use a Qubit hsDNA assay to measure the concentration of barcoded DNA product in each tube. Transfer the DNA product to PCR tubes and store at  prior to library PCR.
-20 °C
Typical concentrations are between 0.5 and 2.0 ng/μl.
?. [Cleanup barcoded DNA and antibody products]
For antibody tag cleanup, add biotinylated capture oligonucleotide to the tube supernatants from Step 26 to a final concentration of .
The capture probe sequence is /5Biosg/GGCTTGTTGTGATTCGACGA/3C6/, using IDT codes.
?. [Cleanup barcoded DNA and antibody products]
Heat the supernatant-probe solution to  for  to denature the PCR product, then snap cool on ice for probe hybridization. Allow tubes to cool for  on ice.
95 °C
?. [Cleanup barcoded DNA and antibody products]
For each sample tube, wash  of magnetic streptavidin beads two times in  D-PBS, allowing beads to bind to the magnet between washes. Resuspend beads in  D-PBS and add to each tube. Pipet to mix.
10 µl
1 mL
10 µl
?. [Cleanup barcoded DNA and antibody products]
Incubate tubes  at  with rotation to allow streptavidin-biotin binding.
95 Room temperature
?. [Cleanup barcoded DNA and antibody products]
Place the tubes on a magnet and allow beads to separate.
?. [Cleanup barcoded DNA and antibody products]
Wash beads two times in  D-PBS and resuspend in 30 μL water. Transfer the bead solutions to PCR tubes and store at  prior to library PCR.
1 mL
-20 °C
?. [Library preparation PCR]
Prepare library PCR reactions for each tube, and for each of the DNA and antibody tag libraries:DNA panel libraries: Mission Bio Barcoding Mix P5 primer () P7 primer () - DNA panel specific of barcoded DNA product in  waterAntibody tag libraries: Mission Bio Barcoding Mix P5 primer () P7 primer () - Antibody tag specific bead-bound antibody tag product
50 µl
25 µl
5 µl
5 µl
4 ng
15 µl
25 µl
5 µl
5 µl
15 µl
Ensure all combinations of DNA panel and antibody tag libraries have unique P5 and P7 barcodes. The DNA panel and antibody tag libraries have different P7 primers, which are listed in the attachment "Supplementary Table 6: Library preparation primer sequences".
?. [Library preparation PCR]
Amplify the DNA panel and antibody tag libraries according to the following protocol, using 10 cycles for the DNA panel and 20 cycles for the antibody tags.
?. [Library cleanup and quantification]
Perform library cleanup for both the DNA panel and antibody tag libraries as described in the attached Mission Bio document (Steps 7.10 to 7.25): "Tapestri Single-Cell DNA AML User Guide". In Step 7.13, use  Ampure XP  beads instead of  .
35 µl
31.5 µl
?. [Library cleanup and quantification]
Quantify the concentration of DNA panel and antibody tag libraries using the Qubit hsDNA assay.
Typical concentrations are between 5 and 20 ng/uL.
?. [Library cleanup and quantification]
Run  of each library on a Bioanalyzer High-Sensitivity DNA chip or comparable TapeStation assay.
1 ng
All libraries should be free of primer-dimers (
?. [Next-generation sequencing]
Pool the libraries for sequencing, the protocol for which varies depending on Illumina sequencing platform. It is most cost-effective to sequence the DNA panel and antibody libraries using separate kits, as the antibody tags require fewer cycles (~80 cycles for antibody tags vs. 300 for DNA panel). A custom Read 1 primer is required in both libraries (see note below).The amount of reads to allot for each library depends on the number of cells sequenced and number of targets in the DNA and antibody panels. As a rule of thumb, allotting 100X coverage for each DNA and antibody target per cell should yield sufficient depth for genotyping and antibody tag counting.
Mission Bio V1 chemistry requires a custom Read 1 primer with the following sequence: GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAG. It should be HPLC purified.
?. [Data processing]
The raw FASTQ files are analyzed by the DAb-seq pipeline available at: https://github.com/AbateLab/DAb-seq. See the README for detailed instructions on setting up and running the pipeline.