GSM9567635: Primary human samples, cell surface proteins for immunophenotype; Patient 4 EOC6; Homo sapiens; OTHER
For DAB-seq, Cryopreserved cells were thawed and normalized to 10,000 cells/μL in 180 μL PBS (Corning). Samples with more than one million viable cells recovered were additionally incubated with Human TruStain FcX (Biolegend) and salmon sperm DNA (Invitrogen) for 15 minutes at 4C. A pool of 9 oligo-conjugated antibodies (CD3, CD4, CD11b, CD13, CD19, CD33, CD34, CD117, CD45)  was added, and cells were incubated for an additional 30 minutes. In addition, individual samples were also incubated with unique hashtags (BioLegend) to provide sample-level identifiers, and groups of 3 individual patients were pooled together in multiplexed runs. Samples were resuspended in Cell Buffer (MissionBio), diluted to 4-7e6 cells/mL, and loaded onto a microfluidics cartridge, where individual cells were encapsulated, lysed, and barcoded using the Tapestri instrument. DNA from barcoded cells was amplified via PCR using a custom targeted panel that included 288 amplicons across 66 genes associated with acute leukemia generally and known clinical mutations in these samples specifically. For CITEseq, 1-2 million cells were blocked with Human TruStain FcX (BioLegend) and stained with a panel of TotalSeq-C antibodies from the Human Universal Cocktail (1 μg/each, BioLegend) in Cell Buffer (BioLegend) for 20 minutes at 4C. After staining, cells were washed in Staining Buffer, resuspended at 1,000 cells/μL, and immediately processed with the 10x Genomics controller and the Single Cell 5' reagent kit with feature barcoding technology for cell-surface proteins. For DABseq, DNA PCR products were isolated, purified with AmpureXP beads (Beckman Coulter), used as a PCR template for library generation, and then repurified with AmpureXP beads. Protein PCR products (when applicable) were isolated from the supernatant from AmpureXP bead purification via incubation with a 5' Biotin Oligo (ITD). Protein PCR products were then purified using Streptavidin C1 beads (Thermo Fisher Scientific), used as a PCR template for library generation, and then repurified using AmpureXP beads. Both DNA and protein libraries were quantified and assessed for quality via a Qubit fluorometer (Life Technologies) and Bioanalyzer (Agilent Technologies) prior to pooling for sequencing on an Illumina Novaseq. For CITEseeq, 5' gene expression and feature barcoding libraries were constructed using a 10x Genomics library preparation kit. Library quality was assessed using Agilent High Sensitivity DNA kit and Bioanalyzer 2100, and libraries were quantified using dsDNA High-Sensitivity (HS) assay kit (Invitrogen) on Qubit fluorometer before being sequenced on an Illumina Novaseq.