GSM8535428: Colon fibroblast, IBD, crohn's, no strain, donor C1, rep1; Homo sapiens; RNA-Seq
RNA was extracted from Colon Chips at two weeks of culture, after functional monolayer and mucus layer formation. Cells in apical and basal channels were harvested separately with RLT lysis buffer from RNeasy Mini Kit (74106; Qiagen, Hilden, Germany). Channels were washed with 100 µL PBS three times. Fibroblasts were lysed first by quickly pressing and releasing the micropipette plunger at least four times. Lysates were collected in 1.5 mL tubes and stored immediately at -80 °C for RNA sequencing analysis. Subsequently, epithelial cell lysates were collected and processed in a similar manner. High-grade RNA (RIN > 8) was prepared for transcriptomic analysis (Agilent Nano Kit (Agilent 2100 Bioanalyzer, Agilent Technologies). Total RNA was isolated from each sample and mRNA was enriched by poly(A) tail selection. Purified mRNA was converted into cDNA libraries and amplified via PCR to increase library concentration.