GSM6865785: Ctrl1Day; Mus musculus; RNA-Seq
Intestinal epithelial cell isolation: The whole intestine was removed and placed into Ca2+/Mg2+-free Hank's buffered salt solution (CMF-HBSS, Thermo Fisher). For jejunum samples, 10 cm was isolated at the midpoint of the small intestine. The intestine was opened longitudinally and vigorously washed in CMF-HBSS. Samples were then cut into 1-2cm pieces, transferred to CMF-HBSS containing 30 mM EDTA, and rotated for 30 min on ice. Samples were shaken vigorously by hand for 5min, passed over a 70 µm filter and crypts were palleted after centrifuge. Crypts were incubated in TryPLE Express  (Thermo Fisher) containing 0.5mM N-acetyl cysteine ( NAC, Sigma), 10uM Y-27632 dihydrochloride ( Y-27632, Sigma) in a 37ºC water bath with stirring by a P1000 pipette for 20 min. The dissociated samples were transferred to Basal Medium (advanced DMEM/F12 medium containing 1X GlutaMAX, 10 mM HEPES, 1X penicillin/streptomycin (all Thermo Fisher), with 5 mM NAC). 50ml tube was shaken for about 30sec to further promote the dissociation. The supernatant was removed after centrifuge and the pellet was resuspended in 3ml media. 20μm syringe filter was used to separate any remaining mass. Dead cell removal kit (Miltenyi Biotec) was then applied to get live single intestinal epithelial cells.  Nuclei isloation: Single-nucleus suspensions were isolated from ~50 mg fresh mouse intestine using Singulator 100 (genomics) according to the manufacturer's protocol. ScRNA-seq: Cells were loaded on a Chromium Single Cell Controller (10x Genomics, Pleasanton, CA), based on live cell concentration assessed by Luna-FL cell counter (Logos Biosystems). Libraries were prepared using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics) according to the manufacturer's directions. ScATAC-seq: High-quality nuclei suspensions were processed to create libraries according to the manufacturer's instructions for the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics) using a Chromium Single Cell Controller.