GSM2474996: PEA1; Homo sapiens; RNA-Seq
Total RNA was extracted from 40 epithelial ovarian cancer cell lines and one immortalized line derived from ovarian surface epithelium (OE-E6/E7) using RNeasy plus mini kit (QIAGEN, 74134). mRNA was quantified using QubitRNA (Invitrogen) and quality control was assessed with bioanalyzer total RNA nano chip. One μg of mRNA was fragmented to an average length of 200 bp by incubation for 5 minutes at 94°C with 5X fragmentation buffer (Illumina, RS-100-0801). Efficiency of the fragmentation was defined on Bioanalyzer RNA Pico Chip. The fragmented mRNA was randomly primed and reverse transcribed using Super Script II cDNA synthesis kit (Invitrogen, 18064-014). After second-strand synthesis, the cDNA went through end-repair and ligation reactions according to the Illumina mRNA-Seq Sample Prep Kit protocol. The cDNA library was size-fractioned on a 2% TBE agarose gel. Material in the 350-400 bp range was excised and purified (Zymo Research, D4001). Half of the eluted cDNA library was used as a template for amplification according mRNA-Seq Sample Prep Kit protocol. The PCR product was purified using the PureLink PCR micro purification kit (Invitrogen, K310050). The library size (350-400 bp) was validated on a Bioanalyzer DNA 1000 Chip and the concentration was estimated using Qubit fluorometer and Quant-iT dsDNA BR Assay Kit (Invitrogen, Q32850). The library was then used to build clusters on the Illumina flow cell and analysis was done using Illumina Hiseq 2000. RNA libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM2474996