GSM4932597: MB231ML162.2; Homo sapiens; RNA-Seq
Three biological replicate tung-oil or control MDA-MB-231 xenograft tumor samples were homogenized in ice-cold PBS using a dounce homogenizer and total RNA was isolated using the RNeasy kit (Qiagen). The stranded mRNA-seq library was generated from 1000 ng of total RNA from each sample using Truseq stranded mRNA library kit (Illumina) according to the product instructions. In short, mRNAs were enriched twice via poly-T-based RNA purification beads, and subjected to fragmentation at 94 C for 8 minutes via the divalent cation method. The first strand cDNA was synthesized by SuperscriptII (ThermoFisher Scientific) and random primers at 42 C for 15 mins, followed by second strand synthesis at 16 C for 1 hour. During second strand synthesis, the dUTP was used to replace dTTP, thereby the second strand was quenched during amplification. A single 'A' nucleotide is added to the 3' ends of the blunt fragments at 37 C for 30 minutes. Adapters with illuminaP5, P7 sequences as well as indices were ligated to the cDNA fragment at 30 C for 10 minutes. After Ampure bead (BD) purification (Beckman Coulter), a 15-cycle PCR reaction was used to enrich the fragments. PCR was set at 98 C for 10 s, 60 C for 30 s and extended at 72 C for 30 s. Libraries were again purified using AmPure beads, checked for quality check with a Bioanalyzer (Agilent Technologies) and quantified with Qubit (Invitrogen). Sample libraries were subsequently pooled and loaded to the HiSeq2500 and sequenced using a Hiseq rapid SRcluster kit and HiSeq rapid SBS kit (Illumina). Single 50bp were generated for the bioinformatic analysis.