GSM7466937: OVCAR5CDDPsshGPX44249_2; Homo sapiens; RNA-Seq
Total RNA was extracted with TRI Reagent (Sigma, Cat# T9424). DNA contamination in the samples was removed by using a RNeasy MinElute Cleanup Kit (QIAGEN) with RNase-Free DNase Set (QIAGEN). mRNA was isolated from 1 µg of total RNA with a NEBNext Poly(A) mRNA Magnetic Isolation Module, which was used for preparing RNA sequencing libraries with the NEBNext Ultra II RNA library prep kit and protocol from Illumina (New England Biolabs Inc., Ipswich, MA). Library quality was verified using a BioAnalyzer (Agilent Technologies), and then sequenced on an Illumina NextSeq500 system with single-end, 75-bp read length settings. For quality control, raw fastq files were pre-processed using TrimGalore (0.4.4) and cutadapt (1.14) with single-end trimming mode, Phred score cutoff of 20 and minimum sequence length cutoff of 20 bp (62). After checking quality with the FastQC tool, raw sequencing reads were aligned to human genome build hg38 using STAR v.2.5.2 (https://github.com/alexdobin/STAR) with standard settings. Mapped reads were converted to raw counts with HTSeq (https://htseq.readthedocs.io/en/master/), normalized to library size, and analyzed for DEGs by edgeR (Bioconductor). The log2(fold-change) andP-value of total normalized counts and DEG counts were then analyzed by GSEA, Ingenuity Pathway Analysis (IPA, QIAGEN). Data are deposited in the NCBI gene expression omnibus