title: GSM8774601: NEC pups_Intestinal epithelium_scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: IECs were isolated from small intestines by washing twice with gut wash buffer composed of HBSS supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 20 mM HEPES, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2% fetal bovine serum (FBS). Tissues were then cut into 1- 2 cm segments and were incubated with gut wash buffer containing 2 mM dithiothreitol at 37℃ for 10 min. Subsequently, the tissues were vigorously shaken and incubated with fresh gut wash buffer containing 5 mM EDTA at 37℃ for 7 min. The fractions containing EDTA were collected and tissues were subsequently incubated with fresh EDTA for extra 7 min. The two EDTA-containing fractions were filtered through 100-μm filters and centrifuged at 300 g for 5 min. Cell pellets were then digested with Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.1 U/mL collagenase I (Invitrogen, USA) and 0.1 mg/mL DNase I (Solarbio) for 8 min in 37℃ water bath. The digestion process was neutralized with 2.5% FBS-DMEM, and the pellets were washed twice with gut wash buffer. Library was performed according to the manufacter's instructions (single cell 3'v2 protocol. 10x Genomics)