title: GSM9022180: Rgs14 KO, sample 2, GEX; Mus musculus; OTHER
library_name: GSM9022180
library_construction_protocol: The colon was isolated from the mouse, opened longitudinally, and cleaned with PBS to remove feces and debris from the lumen. The intestinal epithelium was isolated by incubation in PBS/5 mM EDTA and shaking for 10 min at 37˚C , repeated at least two times, and supernatants were collected and pelleted. After the epithelium was removed, the colon was twice washed, vortexed, and then incubated in PBS/EDTA for 10 min. The colon was then cut as small as possible and digested with RPMI 1640 containing 0.5 mg/ml Collagenase VIII (Sigma-Aldrich #C2139) and 10 U/ml DNase I (Roche #4536282001) for 15 min at 37 ˚C in a water bath with 200 rpm shaking and vortexed every 5 min. Supernatants were collected and passed through a 70-μm cell strainer, and the cells were pelleted by centrifuging at 600 x g for 5 min at 4 ˚C to receive the lamina propria cells. After counting the cells, they were further processed for further treatment, hashtag antibodies labeled-colonic lamina propria monocytes and macrophages were isolated and stained for CCR2, Ly6c, MHC II, CD11b, and CD64. After gating on CD11b+cells, the mononuclear cells were distinguished into CCR2+ or CD64+ mononuclear cells Hashtag antibodies labeled-cell suspension volumes aiming at a targeted recovery of 20,000 cells were loaded into wells of a single 10x Genomics Chromium Single Cell Controller (one well per two biological replicates). Chromium Next GEM Single Cell 3' HT Reagent Kits v3.1 were used according to the manufacturer's instructions for single-cell capture, cDNA, and library preparation. Single-cell libraries were then sequenced on one flow-cell of an Illumina NovaSeq 6000 sequencer machine at the Genomics Facility Basel of the ETH Zurich (with 91nt-long R2 reads).