GSM6784421: Biliary epithelial organoids, Wild-type 2; Mus musculus; RNA-Seq
Once organoids reached confluence (~3 days after first passage), the organoids were extracted with Cell Recovery Solution (Corning, Tewksbury, MA). RNA was then extracted utilizing the RNeasy micro kit (Qiagen, Redwood, CA). Total RNA was isolated by Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. RNA purity was assessed by the 260/280 and 260/230 nm absorbance ratio of 2 or greater. 1 µg of total RNA was used for cDNA synthesis using the Bio-Rad iScript cDNA synthesis kit (Bio-Rad Life Science, Hercules, CA). Analysis of gene expression via Reverse-Transcription Polymerase Chain Reaction (RTPCR) was performed on Bio-Rad C1000 Thermal cycler using Taq PCR Master Mix Kit (Qiagen, Valencia, CA) and intron spanning gene specific primers (Table S2). Quantitative Real-Time PCR (qPCR) was performed using cDNA using Light-Cycler Taqman Master (Roche Applied Science, Indianapolis, IN) and probes from the Universal Probe Library (Roche Applied Science) using intron spanning, gene specific primers. Relative expression levels were calculated by Δ–Δ Ct method. Actin was used to normalize the gene expression.