title: GSM8898144: induced pluripotent stem cell-derived fibroblasts grown in intestinal cell medium, replicate 3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from iFBs. Briefly, models were lysed in PureLink RNA Mini Kit lysis buffer. RNA was isolated according to the manufacturer's protocol. Total RNA was quantified, and cDNA was synthesised, using iScriptTM cDNA Synthesis Kit. The subsequent RT-qPCR was performed using SYBR Green I Master Mix (Bio-Rad Laboratories). 18S served as a Housekeeping gene control. For library preparation, we used the Novogene NGS RNA Library Prep Set (PT042). Polyadenylated mRNA was enriched with oligo(dT)25 beads, randomly fragmented, and reverse-transcribed using random hexamers. The resulting double-stranded cDNA underwent purification, end-repair, A-tailing, and adapter ligation before PCR enrichment with indexed P5 and P7 primers. After quality checks via Qubit 2.0, real-time PCR, and Bioanalyzer 2100, libraries were pooled and sequenced on the Illumina Novaseq X platform (paired-end 150, PE150).