title: GSM9038865: preInfusion, VDJ, 2; Homo sapiens; RNA-Seq
library_construction_protocol: Leftover clinical anti-CD19 CAR T cells (Kymriah, Novartis) of three donors were rinsed from the infusion bag and tubes and peripheral blood mononuclear cell (PBMC) were isolated and then cryopreserved. Without rest, thawed CAR-T cells and Nalm6 cells were washed in medium (in RPMI 1640 + 10% FCS + 2 mM L-glutamine + 25 mM HEPES + 20 IU/mL IL-2). After manual counting, 2 mL CAR T cells of each donor (5x10^5 cells/mL) were either co-cultured with 0.5 mL Nalm6 (1x10^6 cells/mL) (=stimulated) or 0.5 mL of additional medium(=unstimulated), respectively, in 6-well plates at 37°C, 5% CO2 for 24 h. These stimulated and unstimulated pre-infusion CAR-T cells were subjected to flow-based cell sorting. After an initial washing step with PBS (300 xg for 6 min at 21°C), samples were incubated with ZombieAqua (1:100, BioLegend) for 10 min in the dark at room temperature. Samples were washed once with 1 mL PBA (PBS + 0.25% BSA + 0.02% NaN3) and human CD19 CAR Detection Reagent (1:50, Milentyi biotec, 130-129-550) were added, mixed and incubated for 10 min at room temperature in the dark. After washing twice with 2 mL PBA at room temperature, an antibody mastermix comprising anti-CD19-PE antibody (1:50, clone 4G7, BioLegend), anti-CD3-APC (1:33, UCHT1, BioLegend) and anti-Streptavidin-PE/TexasRed (1:200, ThermoFischer, SA1017) was added and incubated for 20 min at room temperature in the dark, followed by two final washing steps in sorting buffer (PBA + 1 mM EDTA). Single, live CD19- CD3+ lymphocytes (8333 cells per donor and condition) were sorted on a MoFlo XDP (Beckman Coulter) sorter. Immediately after sorting, cells were pooled into one 10X sample according to their stimulation condition and loaded to a Chromium Next GEM Chip K (10X Genomics) and Chromium Next GEM Single-Cell 5′ kits v.2 were used to generate GEX and VDJ libraries according to the manufacturer's instructions (10X Genomics, 1000266, 1000196, 1000256, 1000252, 1000287, 3000431, 3000510). Libraries were sent to Novogene and sequenced on an Illumina NovaSeq platform with PE150 strategy. Library construction was performed according to manufacturer's instructions (10x Genomics) for 5' GEX and VDJ, V2 chemistry dual index
identifiers.namespace: GEO
identifiers.id: GSM9038865_r1