GSM8386414: colon, WT2; Mus musculus; RNA-Seq
Following the removal of the mesentery/fat and connective tissue, colons from three mice were combined, flushed with ice-cold CMF-PBS, longitudinally opened, and rinsed with ice-cold CMF-PBS. The colons were then cut into 0.5-1 centimeter pieces and washed once in rinse buffer containing 5.6 mM Na2HPO4 x2H2O, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM D-sorbitol. Then, the colon pieces were resuspended in isolation buffer (2mM EDTA and 0.5mM DTT in rinse buffer) and incubated on a rotation platform for 20 min at room temperature. Subsequently, supernatants were removed and the remaining tissue was resuspended in 10 ml ice-cold rinse buffer and pipetted 25 times up and down with a 25 ml pipette. Following the settling of the large tissue pieces, the supernatants containing crypts were transferred to a new tube.  After additional pipetting 25 times up and down with a 25 ml pipette and settling of the tissue pieces, the supernatants were centrifuged at 300 g, 4°C for 3 min, and the pellets were resuspended in 10 ml room-temperature single-cell-isolation-buffer (1 mg/ml DNaseI, 5 mM MgCl2, 20 μM Y27 in TrypLE Express) and incubated on 100 RPM at 37°C for 20 min. Then,  the cell solution was passed through the 40 mm cell strainers. Digestion was stopped by the addition of 35 ml ice-cold CMF-PBS, and centrifugation at 500 g, 4°C for 4 min. The cell pellets were resuspended in ice-cold PBS with 2% FBS. Single cell libraries were prepared on a 10x Genomics Chromium controller (10x Genomics, Pleasanton, CA) using the version 3 reagent kit according to the manufacturer's instructions. The reagent mixture was calculated for capturing 6000 cells. The libraries were sequenced on a NovaSeq (Illumina, San Diego, CA) by the NIEHS Epigenomics core laboratory. Raw read processing was carried out using the Cell Ranger Single-Cell Software Suite (version 3.0.1, 10X Genomics).