title: GSM7866652: 4h_HTO; Homo sapiens; OTHER
library_construction_protocol: Peripheral blood mononuclear cells (PBMCs) were isolated from 500 ml whole human blood obtained from the New York Blood Center as described previously10. Briefly, whole blood was processed within 2h of receipt and centrifuged at 480 g for 10 min. at room temperature (RT). Plasma was collected and the cell fraction diluted 1:2 in PBS, then placed in Ficoll-Paque PLUS density gradient media (GE Healthcare Life Sciences, 17144003) and centrifuged at 1,250 g for 20 min. at RT. The PBMC layer was collected, washed twice in PBS and resuspended at 2–5×105 cells per ml in 50% vol/vol RPMI-1640 (Gibco, 11875093) 40% vol/vol FBS 10% dimethyl sulfoxide (Tocris Bioscience, 3176) for frozen storage at –80 °C or liquid N2. On experiment date, PBMCs were thawed and placed in RPMI-1640 supplemented with 10% vol/vol FBS and 100 μg ml−1 penicillin-streptomycin (Corning, MT30002CI) to acclimatize in a humidified incubator at 37 °C and 5% CO2 for 2h. Viable PBMCs were cultured at 5×105 cells per well of a 12-well plate coated with Dynabeads Human T-Activator CD3/CD28 (Gibco, 11161D) to activate T cells. Following acclimatization and activation, 1 μg ml−1 anti-PD-1 (SIM0003, Bio X Cell) or 20 μg ml−1 anti-CTLA4 (SIM0004, Bio X Cell) were added to the corresponding experimental wells for 4h or 24h. Next, samples were washed in phosphate-buffered saline (PBS; Corning, 1-031-CV), centrifuged for 5 min. at 300 g and placed in PBS supplemented with 1% vol/vol bovine serum albumin (BSA, Jackson ImmunoResearch, 149856) and 10 μg Anti-Fc Receptor Binding Inhibitor (Invitrogen, 14-9161-73) for 10 min at 4 °C to prevent nonspecific binding in later steps. Fc-blocked samples were incubated with 0.5 μg unique hashtag antibodies from the 3ʹ CellPlex Kit Set A (10x Genomics, PN-1000261), respectively, and stained with the TotalSeq™-B Human Universal Cocktail, V1.0 (BioLegend, 399904) according to the manufacturer's instructions. Stained, viable cells were submitted to the Genome Technology Center at the NYU Grossman School of Medicine at NYU Langone Health (NYU GTC) for CITE-seq analysis following the Single Cell 3' v2 workflow and using a 3' CellPlex Kit, Set A for multiplexing (10x Genomics, 1000261) through the cDNA amplification step. The transcriptome library was generated according to the protocol by Stoeckius M. et al.82 Separate PCRs were used to generate the CITE-seq antibody-derived tag (ADT) protein library (SI-PCR and RPI-x primers) and the hashtag Iibrary, respectively. Finally, the ADT and hashtag libraries were sequenced on an Illumina NovaSeq 6000 using the S4 200 cycle flow cell v1.5, also at the NYU GTC.