RNA-seq of Limonoid-sensitive and -insensitive cell lines
Three biological replicates of MEL-HO wild-type and MEL-HO cyp27a1-/- were treated with DMSO, IC20, and IC70 of Limonoid (0.1% DMSO vol/vol) for 24 hours, 48 hours, and 72 hours. Total RNA was isolated according to the manufacturer’s protocol with an RNeasy kit with on-column DNA digestion (Qiagen). RNA concentration and integrity was measured by fragment analyzer with Standard Sensitivity RNA kit according to the manufacturer’s protocol (Advanced Analytical Technologies). RNA-seq libraries were prepared using the TruSeq Stranded mRNA Sample Preparation kit (Illumina) and sequenced in strand specific paired-end mode, 2x76bp, using the HiSeq2500 platform.