GSM6965397: Tumor, No-RT hypoxia, late, biol rep 1; Homo sapiens; RNA-Seq
Tumors were sampled 24h after radiotherapy and were disrupted in cold RLT lysis buffer with automated homogenizer (TissueLyser II, Qiagen) and total RNA extracted using the RNEasy mini kit (Qiagen) according to manufacturer instructions. RNA quality (RQN > 6.8) was assessed on a Fragment Analyzer (Agilent Technologies). RNA-seq libraries were prepared from 500 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina) using a unique dual indexing strategy, and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorimetric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies). Cluster generation was performed with 2 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents and sequenced on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 150 cycles (single end).