title: GSM7115904: Rectum, responder post Vedo treatment 22R_Rectum ADT; Homo sapiens; OTHER
library_construction_protocol: Four intestinal biopsies were obtained with endoscopic biopsy forceps from the ileum and rectum and collected in separate conical tubes with Hank's buffered saline solution (HBSS, Corning). 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, Thermo Fisher Scientific) for 10 minutes on a rocker at 37°C, followed by a final HBSS wash. Intestinal biopsies were mechanically dissociated, then placed into a conical tube containing 10 mL of digestion mixture [complete RPMI + 1.5 mg/mL Collagenase type VIII (Sigma-Aldrich) + 50 µg/mL DNase I (Roche)]. The biopsies were digested in a shaking incubator (225 rpm) for 20 minutes at 37°C. The reaction was stopped with phosphatebuffered saline (PBS) pre-cooled to 4°C, filtered, and resuspended in fresh complete RPMI. Samples were incubated with BD Pharmingen Human BD Fc Block for 10 minutes to block non-specific binding of the Fc receptor as described in the manufacturer's protocol. The 55-plex BD AbSeq antibody cocktail was generated using 2.6 uL of each antibody and brought to volume with BD buffer. Concurrently, samples were stained with anti-human CD45 and a live/dead stain, and incubated for 60 minutes on ice. CD45 + immune cells were sorted on a FACSAria2 and collected in complete RPMI media with 10% FBS. 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) Dimethylsulfoxide (DMSO, Sigma Aldrich), 40%(v/v) complete RPMI [RPMI (Corning) +10%(v/v) fetal bovine serum (FBS, Life Technologies) +100 U/mL penicillin/100 µg/mL streptomycin (Life Technologies)], 50%(v/v) FBS), placed into a freezing container (Mr. Frosty), and stored at -80°C. Cells were recovered, washed, filtered, and labeled with anti-human CD45 (2D1) (Biolegend) for sorting. CD45 + immune cells were sorted on a FACSAria2 (BD Biosciences). Cells were washed and resuspended in phosphate-buffered saline (PBS) + 0.04%(w/v) bovine serum albumin (UltraPure BSA, Thermo Fisher Scientific) per the manufacturer's guidelines. Single-cell libraries were prepared according to the protocol for 10x Genomics for Single Cell 3' Gene Expression. Approximately 20,000 sorted CD45 + cells resuspended at a final concentration of 4,000 cells/uL were loaded and partitioned into Gel Bead-in-EMulsions (GEMs). The 10x Genomics cDNA generation protocol was modified to include the BD AbSeq PCR1 primer. After PCR amplification, the fractions containing the AbSeq oligos were processed through a separate library construction workflow. This AbSeq protocol generates libraries directly after cDNA amplification, eliminating intermediate steps as described in the 10x Genomics protocol. The quality of the sample fragments was determined by the High Sensitivity D5000 and D1000 ScreenTape assay (Agilent Technologies). The scRNA and protein libraries were pooled at equimolar amounts for a final 10nM concentration and sequenced on a NovaSeq 6000 S4 flow cell (Illumina).