title: GSM4801624: 30IEC_CL_D3_4; Mus musculus; RNA-Seq
library_construction_protocol: Epithelial cells were isolated from large intestines after flushing with ice-cold sterile PBS. The entire length of the colon was opened longitudinally and washed with Ca2+/Mg2+-free HBSS supplemented with 2% bovine serum albumin and 10 mM HEPES. The colon was then cut into small pieces (5-10 mm) and immersed in 20 ml of Ca2+/Mg2+-free HBSS with 2% fetal calf serum, 1 mM EDTA, 10 mM HEPES, and 1 mM dithiothreitol, and incubated at 37°C at 200 rpm for 20 minutes and the process was repeated twice. The three fractions of epithelial cells were pooled, passed through a 100 μM mesh nylon filter, and centrifuged at 1,000 g for 5 minutes at 4°C. Lymphocytes and other CD45+ cells were removed from the epithelial cell fraction by overlaying onto a 40-90% Percoll gradient followed by centrifugation at 2,200 rpm for 20 minutes. The remaining epithelial cells were collected and washed with ice-cold HBSS and centrifuged for 1,000 g for 5 minutes at 4°C. The final pellet was then collected as colonocytes, and purity was assessed by flow cytometry using anti-CD45, anti-EpCAM, and anti-cytokeratin-18 antibodies. RNA libraries were prepared for sequencing using TruSeq stranded mRNA library prep kit following standard Illumina protocols The pooled libraries were sequenced on NextSeq 500 Illumina sequencer using NextSeq 500/550 High Output Kit v2.5 (75 cycles) (Illumina #20024906) in a single read mode.