GSM9514668: scRNA-seq+ TCR-seq+BCR-seq (Multiplexed Samples S25A-S48A, S25B-S48B); Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMC) were isolated from whole blood. In brief, blood samples (5-10 mL) were poured into Accuspin tubes (Sigma-Aldrich, Catalog No. A2055) layered with Ficoll (GE Healthcare, Catalog No. 14-1440-03,) and centrifuged to generate a buffy coat layer. PBMCs were isolated from the buffy coat layer, concentrated, and placed in Gibco Recovery™ Cell Culture Freezing Medium (Catalog No. 12648010). Samples were kept in Mr. Frosty containers in -80C freezer and subsequently stored in liquid nitrogen until downstream experiments. The BD® Human Single-Cell Multiplexing Kit (Catalog No. 633781) was used to multiplex PBMCs from each patient in batches of 12 patients. About 5,000-10,000 multiplexed PBMCs from each patient were loaded into the BD Rhapsody™ Cartridge (BD Biosciences, Catalog No. 633733). Single-cell capture, barcoding, lysis, and cDNA synthesis were performed with the BD Rhapsody™ Express Single-Cell Analysis System according to the manufacturer's instructions. Paired TCR/BCR Full Length (BD Rhapsody™ TCR/BCR Amplification Kit, Catalog No. 665345) and mRNA Whole Transcriptome Analysis (WTA) (BD Rhapsody™ WTA Amplification Kit, Catalog No. 633801) libraries were indexed according to manufacturer's instructions. Libraries were sequenced using the NovaSeq X Plus sequencer at a depth of 25,000 reads for WTA and 5,000 reads per cell for TCR and BCR.