title: GSM8397701: HOM_2; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated using the phenol-chloroform method (Chirgwin et al., 1979). Total RNA was then treated with DNase I (DNA-free DNase Treatment & Removal Reagents, Ambion) according to manufacturerʼs instructions. Total RNA samples (500ng; RQS/RIN average 5.6, range 4.7-6.5) were converted into cDNA sequencing libraries with the "QuantSeq 3' mRNA-Seq Library Prep Kit (FWD) for Illumina" (Lexogen, Cat.No. 015). Briefly, library generation is initiated by reverse transcription with oligodT priming, followed by a random-primed second strand synthesis. Primers from both steps contain Illumina-compatible sequences. Libraries were completed by PCR, and sequenced on an Illumina instrument (see below) by following manufacturer's protocols. {This library preparation kit generates directional libraries stranded in the sense orientation (the read1 (the only read in single read format) has the sense orientation (--library-type fr-secondstrand in TopHat, --stranded=yes in HTSeq)}