GSM5176233: CAR T cells scRNAseq; Homo sapiens; RNA-Seq
Cryopreserved patient biospecimens were thawed in warm RPMI+10% FBS. Cells were first washed with cold FACS buffer (PBS, 2% BSA, 0.05% sodium azide). Next, Fc receptors were blocked by incubation with Human TruStain FcX (BioLegend, 422301) at 1:50 dilution for 5 minutes at 4°C. Then, cells were incubated for 30 minutes at 4°C in the dark with a staining solution containing BV421-anti-CD3E (clone SK7) and Alexa Fluor 647-labeled antigen-tetramers at 3 nM final concentration for CAR-detection. Subsequently, cells were incubated with LIVE/DEAD Fixable Near-IR viability dye (Invitrogen, L34975) diluted 1:1000 in PBS for 5 minutes at room temperature. Finally, cells were washed three times in cold cell media before fluorescence-activated cell sorting. To draw sorting gates based on a biological control, cells from a healthy donor were similar stained and analyzed 12,000 sorted endogenous CAR- T cells and 7,853 sorted CAR+ T cells were  separately partitioned into droplets for single-cell omics assays via Chromium Next GEM Single-Cell 5'Kit v2 (10x Genomics, 1000263). From the resulting barcoded nucleic acids, TCR-seq libraries were prepared via the Chromium Single-Cell Human TCR Amplification Kit (10x Genomics, 1000252). Sequencing libraries were quantified via the Qubit dsDNA HS Assay Kit (Invitrogen, Q32851), quality-checked for fragment sizes via a high-sensitivity D5000 screentape (Agilent, 5067-5592), pooled, and sequenced (Illumina, NovaSeq-6000).