GSM6281744: 1307_GA_Neg; Homo sapiens; RNA-Seq
After 24 hrs, cells were resuspended in PBS with 10% (v/v) FBS and incubated at 4C for 10 min. Cells were then stained with fixable viability dye eFluor506 (eBioscience) and an antibody mixture containing anti-human CD3-AF700 (UCHT1; ThermoFisher), CD4-APCeFluor780 (RPA-T4; eBioscience), CD8-BV650 (RPA-T8; BioLegend), CCR7-PerCPCy5.5 (G043H7, Biolegend) CD14-V500 (M5E2; BD Bioscience), CD19-V500 (HIB19; BD Bioscience), CD25-FITC (M-A251; BD Bioscience), CD45RA-eFluor450 (HI100, eBiosciences), CD137-APC (4B4-1; BioLegend), OX40-PECy7 (Ber-ACT35; BioLegend), and PD-L1-PE (29E.2A3; BioLegend) for 20 min at room temperature. Cells were washed and resuspended in PBS before being transferred into a 5 ml polypropylene FACS tube (BD Bioscience). PBMCs were sorted, based on forward and side scatter excluding debris and doublets, on a FACSAria into TRIzol LS (Thermo Fisher). Acquisition files were analyzed using FlowJo. Total RNA was extracted from ~100,000 cells in TRIzol LS using the miRNeasy Micro Kit (Qiagen) on a QIAcube (Qiagen). Total RNA was amplified according to Smart Seq protocol (2). cDNA was purified using AMPure XP beads. cDNA was used to prepare a standard barcoded sequencing library (Illumina). Samples were sequenced using an Illumina HiSeq2500 to obtain 50-bp single end reads. Samples that failed to be sequenced due to limited sample availability or failed the quality control were eliminated from further sequencing and analysis. The full protocol can be found at protocols.io (http://dx.doi.org/10.17504/protocols.io.bxr6pm9e). Sequencing was performed according to a paired-end strategy using the NovaSeq sequencer (Illumina). Samples that failed quality control standards were eliminated from further analysis21.