title: GSM5472021: MCF7-siHuR-Doxo-n1; Homo sapiens; RNA-Seq
library_construction_protocol: RNA from whole cells was extracted with TRIzol Reagent (ThermoFischer Scientific) according to the manufacturer's instructions, and 1 µl of GlycoBlue (ThermoFisher Scientific) was added for RNA precipitation. RNA was treated with TURBO DNase I (ThermoFisher Scientific) and quantified using a Nanodrop 2000 spectrophotometer (ThermoFischer Scientific). For sequencing, RNA samples were analyzed using an RNA 2100 Bioanalyzer (Agilent). Total RNA was subjected to DNAse I treatment. 500 ng of good quality RNA (RIN > 9) were used for Illumina compatible library preparation using the TruSeq Stranded total RNA protocol allowing to take into account strand information. A first step of ribosomal RNA depletion was performed using the RiboZero Gold kit (Illumina). 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. 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 100 million clusters (200 million raw paired-end reads) per sample.