title: GSM7508780: Hgfac WT day2 wound_1; Mus musculus; RNA-Seq
library_construction_protocol: Day2 colonic wounds or intact tissue samples from Hgfac WT and Hgfac KO mice were used. Endoscopic wounds were created by the biopsy forceps on day0 and excised by a 2 mm skin biopsy punch tool under the dissecting microscope on day2. Three wounds per mouse were pooled as a sample. Intact tissues excised by a 2 mm skin biopsy punch tool were also collected and used as an unwounded sample. Epithelial and IEL fractions were isolated from wound biopsies or intact colons via mechanical disruption. Briefly, tissues were disrupted via centrifugal force generated by a small magnet in a flask at 1,100 rpm for 45 min at 37°C in 4 ml of RPMI 1640 medium supplemented with 2 mM EDTA. The supernatant was used for epithelial and IEL fractions and the remaining tissues were used for the lamina proprial fraction. For epithelial and IEL fractions, the supernatant was collected and was centrifuged at 16,000 rpm for 8 min. The pellet was suspended with Advanced DMEM/F12 containing 10% FBS medium once and was centrifuged at 16,000 rpm for 8 min. After removing the supernatant, the pellets were suspended with 1 ml of TripLE at 37°C for 10 min. After washing with Advanced DMEM/F12 containing 10% FBS, the pellet was suspended with FACS buffer and kept on ice during lamina proprial isolation. For the lamina proprial (LP) fraction, the tissues were digested in media containing 20% FBS in RPMI 1640 with 0.5 mg/ml of Collagenase VIII (Sigma) for 60 min. The supernatant was collected, filtered, and centrifuged at 16,000 rpm for 8 min. After washing with FACS buffer (2% FBS in PBS) once, cells were used for the analysis. Finally, both epithelial and IEL fraction and LP fraction were combined and used for scRNA-seq. Library was performed according to the manufacturer's instructions (single cell3' v3 protocol, 10x Genomics)