GSM5454354: CTL2; Mus musculus; RNA-Seq
Total RNA from cisplatin exposed (n=2) and non-treated controls (n=2) HEI-OC1 cells was extracted using TRIzol (Thermo Fisher Scientific, USA), and purified by column using RNeasy Mini kit (QIAGEN). Samples (1 µg total RNA per sample) were shipped to Novogene (California, USA) for RNA sequencing. The mRNA from Eukaryote organisms is purified from total RNA using poly-T oligo-attached magnetic beads. The mRNA is first fragmented randomly by addition of fragmentation buffer. First strand cDNA is synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis is subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA is purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments are purified with AMPure XP system (Beckman Coulter, Beverly, USA). Finally, the final library is enriched by PCR amplification and purification of PCR products by AMPure XP beads. Library quality construction: After library construction, diluting library to 1.5 ng/ul with the preliminary quantitative result by Qubit2.0 and detecting the insert size by Agilent 2100. Q-PCR is used to accurately quantify the library effective concentration (> 2nM), in order to ensure the library quality. Libraries are fed into Illumina machines after pooling according to activity and expected data volume if library quality is up to standard.