title: GSM3443112: D038-D029T05: EGF 72 replicate 2; Homo sapiens; RNA-Seq
library_construction_protocol: MDA-MB-468 cells were grown to ~ 70% confluence on 140 cm2 dishes and treated with EGF (100 ng mL-1) for 72 h in biological duplicate. Untreated cells were used as an appropriate control. Whole cell extracts (~ 9 x 105 cells) were collected by centrifugation at 500 x g for 5 min at 4ºC and washed twice in ice-cold PBS1x. Total RNA were extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol and subjected to DNase I treatment using the RNase-free DNase set (Qiagen).  The total RNA was then analyzed using Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer System to determine RNA concentration, qualify RNA Integrity Number (RIN) which was high with RIN = 10 for all samples and confirm removal of DNA contamination.  After quality control of RNA, the ERCC RNA Spike-In (Ambion) was added to RNA samples (RNA samples: RNA Spike-In control diluted at 1/100, 5:1). RNA sequencing libraries were prepared from 1µg of total RNA using the Illumina TruSeq Stranded mRNA Library preparation kit which allows to perform a strand specific sequencing. A first step of polyA selection using magnetic beads is done to focus sequencing on polyadenylated transcripts. After fragmentation, cDNA synthesis was performed and resulting fragments were used for dA-tailing followed by ligation of TruSeq indexed adapters. PCR amplification was finally achieved to generate the final barcoded cDNA libraries. The 4 libraries were equimolarly pooled and subjected to qPCR quantification using the KAPA library quantification kit (Roche). Sequencing was carried out on the NovaSeq 6000 instrument from Illumina based on a 2*100 cycle mode (paired-end reads, 100 bases) using a S1 flow cell in order to obtain around 35 million clusters (70 million raw paired-end reads) per sample