GSM9347813: Colon_DSS_WT; Mus musculus; RNA-Seq
The colon tissues were longitudinally dissected, and cut into 0.5 cm pieces in ice-cold HBSS (w/o) . These tissue pieces were then transferred to 20 mL of pre-digestion solution (5% FBS + HBSS (Ca2+ & Mg2+ free) + 1 mM DTT + 5 mM EDTA + 10 mM HEPES) and shaken horizontally at 37°C for 20 minutes to separate crypts. The digested tissue pieces were collected on a 100 μm MACS filter, fresh pre-digestion solution was added, and the process was repeated twice to further remove intestinal epithelial cells. The tissue pieces were then rinsed with Hanks buffer,and the rinse was followed by digestion in lamina propria digestion solution (RPMI1640, 0.05% type II collagenase, 5% FBS, and DNAase 100 μg/mL) at 37°C for 30 minutes, with the process repeated twice. The digestion solution was filtered through a 70 μm cell filter to obtain the supernatant, which was then centrifuged at 500 g for 8 minutes to obtain cell clumps. Non-cellular debris was removed by Percoll gradient centrifugation, and the single-cell suspension was obtained by filtering through a 40 μm cell filter. cDNA was enzymatically segmented into approximately 200-300 bp fragments. Following this segmentation, standard library preparation steps were undertaken, including the attachment of sequencing adapters and primers. A final round of PCR amplification was conducted to finalize the DNA library.Library construction and 10× single-cell sequencing were performed according to the standard protocol provided by LC-Bio Technology (Hangzhou, China). Briefly, single cells were captured using the 10× Genomics Chromium Single-Cell 3' kit (V3) on a 10× Chromium chip, and cDNA amplification and library construction were performed. The libraries were then sequenced on an Illumina NovaSeq6000 sequencing system by LC-Bio Technology (Hangzhou, China).