GSM5931336: sc5_D14_BCMA_d0 [scRNA-seq]; Homo sapiens; RNA-Seq
FACS-sorted CAR T cells (BFP+ cells) from three independent donors were interrogated using the 10X Chromium platform for simultaneous transcriptome and TCR profiling, with 28,117 cells (range, 7,416-10,5093 cells/donor) passing our quality control (QC) thresholds and taken into subsequent analysis. These cells were sequenced to an average depth of 42,296 reads/cell, yielding an average of 2,541 genes/cell. scRNA-seq was performed using the Chromium Single Cell 5′ Reagent Kit (10X Genomics) according to the manufacturer's instructions. Briefly, CAR T cells were sorted in 100µl 1X PBS, 0.04% BSA and their concentration and viability were determined using Nexcelom's Cellometer K2 Fluorescent Cell Counter. Cell concentration was adjusted to 700 to 1200 cells/µl to maximize the likelihood of achieving the desired cell recovery target. Then single-cell suspension was mixed with RT Master Mix and loaded together with barcoded single-cell 5′ gel beads and partitioning oil into separate single cell A Chips to generate Gel Beads in Emulsion (GEMs) using Chromium Controller. Thus, individual cells were lysed and the polyadenylated RNA was reverse-transcribed and barcoded inside each GEM. Barcoded complementary DNA (cDNA) was recovered through post GEM-RT cleanup and PCR-amplified. cDNA was quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and its profile examined using High Sensitivity D5000 ScreenTape Assay (Agilent Technologies). Fifty nanograms of double-stranded cDNA were used for 5′ Gene Expression library construction. cDNA was fragmented, end-repaired, A-tailed and ligated to an adaptor. A final PCR-amplification with barcoded primers was performed for sample indexing. Single-cell TCR repertoire sequencing. TCRTCR α/β sequencing was performed with 10X Genomics Single Cell V(D)J Immune Profiling Solution (10× Genomics). Briefly, full-length V, D and J gene segments were amplified from barcoded cDNA using a Chromium Single Cell V(D)J Enrichment Kit (human T cells) by means of two sequential PCRs with primers targeting the adaptor and the gene constant region. Enriched cDNA was quantified as described above and 50 ng were further used for library construction as described above. After quality control and quantification, Single Cell V(D)J enriched libraries were pooled and sequenced in an Illumina's HiSeq X (Read1: 150 cycles; Read2: 150 cycles, i7 index: 8 cycles) at an average depth of 5000 reads per cell.