title: GSM3576479: C30_R_10x_TCR; Homo sapiens; OTHER
library_construction_protocol: Human Peripheral Blood Mononuclear Cell Isolation  Blood was collected in BD Vacutainer CPT tube and centrifuged at 400 x g for 25 minutes. The buffy coat layer was removed, washed, and counted. Cells were resuspended in freezing buffer (10%(v/v) DMSO, 40%(v/v) complete RPMI [RPMI +10%(v/v) fetal bovine serum (FBS) +100 U/mL penicillin/100 μg/mL streptomycin], 50%(v/v) FBS), placed into a freezing container (Mr. Frosty), and stored at -80°C. Cells were recovered, washed, and filtered, and labeled with anti-human CD45. CD45+ immune cells were sorted on a FACSAria2 (BD Biosciences).     Human Intestinal Cell Isolation  Four intestinal biopsies were obtained with endoscopic biopsy forceps from the rectum or ileum and collected in a conical tube with Hank's buffered saline solution (HBSS). Intestinal biopsies were transferred into freezing buffer (10%(v/v) DMSO, 40%(v/v) complete RPMI, 50%(v/v) FBS) and stored at -80°C. Biopsies were recovered, incubated in HBSS on a shaker, then incubated twice in HBSS + 5 mM dithiothreitol (DTT) with shaking, then washed in HBSS. Intestinal biopsies were mechanically dissociated, then placed into 10 mL of digestion mixture (complete RPMI + 1.5 mg/mL Collagenase type VIII + 50 µg/mL DNase I) on a rocker at 37°C for 20 minutes, filtered and stained with anti-human CD45. CD45+ immune cells were sorted on a FACSAria2 (BD Biosciences). Single cell libraries were prepared according to the protocol for 10X Genomics for Single Cell V(D)J + 5' Gene Expression. Approximately 20,000 sorted CD45+ cells were loaded and partitioned into Gel Bead In-Emulsions (GEM). scRNA libraries were sequenced on a HiSeq4000 (Illumina). The B and T cell receptor libraries were amplified according to the manufacturer's protocol and sequenced on a NovaSeqS4 (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM3576479