GSM9558946: CAR-T Enterocolitis 4; Homo sapiens; RNA-Seq
Tissue first underwent epithelial layer removal via two incubation steps (20 min, 37°C, rotating incubator 180 rpm) in 10mL of Hanks' balanced salt solution (HBSS), EDTA (0.5 M, pH 8; Invitrogen), Hepes (1 M; Lonza), and fetal bovine serum. Tissue was vortexed, washed with HBSS, and incubated (40 min, 37°C, rotating incubator 180 rpm) in digestion media, containing RPMI, collagenase (Sigma-Aldrich), deoxyribonuclease 1 (Sigma-Aldrich), and fetal bovine serum. Tissue was then manually disrupted an 18-gauge needle and filtered through 100-μm and 40-μm cell strainers, yielding single cell suspension. Sixteen samples (NV = 11, CAR-TCTRL = 2, CAR-TEC = 3) were prepared using 3′ V3 chemistry, and five samples (CAR-TCTRL = 3, CAR-TEC = 2) using 5′ V3 chemistry. Approximately 16,000 cells were loaded per channel for both 3' and 5', targeting a recovery rate of 10,000 cells per sample. Barcoded cDNA was extracted from the GEMs after Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly A-tailing, adapter ligation, and 10x-specific sample indexing following the manufacturer's protocol.