title: GSM6674764: Patient P1, mRNA-derived cDNA; Homo sapiens; OTHER
library_construction_protocol: The frozen PBMCs were quickly thawed at 37°C and gently resuspended by serial additions of DMEM + 10% heat-inactivated FBS, for a final volume of 14ml. Following centrifugation at 300g for 5 min, cells were washed twice with 5ml DMEM + 10% HI-FBS to remove cell debris. Cell counting and viability assessment was done using the LIVE/DEAD™ Viability kit (ThermoFisher Scientific), according to manufacturer's guidelines. Residual red blood cells were removed using 25ul of the antibody cocktail and beads from the StemCell EasySep PBMC isolation kit. CITE-seq antibody labeling: One million cells were resuspended in 1ml Biolegend Cell Staining Buffer and passed through a 40μm Flowmi cell strainer (Sigma) to remove aggregates. Cells were then spun at 400g for 5 min and resuspended in 45ul of Biolegend Cell Staining Buffer complemented with 5ul of Human TruStain FcX Fc block (Biolegend). Cells were then incubated for 30 min at 4°C with a cocktail of TotalSeq-B antibodies (Biolegend) that are DNA-barcoded: CD11c (S-HCL-3), CD141 (M80), CD161 (HP-3G10), CD14 (Me5E2), CD16 (3G8), CD19 (HIB19), CD1c (L161), CD28 (CD28.2), CD370 (CLEC9A) (8F9), CD38 (HB-7), CD3e (UCHT1), CD4 (RPA-T4), CD45RA (HI100), CD45RO (UCHL1), CD56 (NCAM) (5.1H11), CD66b (6/40c), CD69 (FN50), CD8a (RPA-T8), TCR gd (B1), TCR Va7.2 (3C10), IgG1 k Isotype Ctl (MOPC-21). Cells were washes twice in Cell Staining Buffer, once in PBS + 0.04% BSA, and resuspended at a concentration of 1000 cells/μl in PBS + 0.04% BSA. Single-cell library: The samples were loaded onto a 10X Genomics Chromium G chip aiming to capture 10,000 cells per samples. The reverse transcription and library preparation was performed using the Chromium Single Cell 3′ v3.1 Reagent Kits following manufacturers guidelines. The cDNA and feature barcode library quality was assessed using a TapeStation (Agilent). Sequencing: The cDNA and feature barcode libraries were sequenced on S4 flowcells of an Illumina NovaSeq 6000 sequencer.