title: GSM4775005: mESC2R; Mus musculus; RNA-Seq
library_construction_protocol: 20 µg TriZOL purified RNA from each sample was depleted of rRNA using a Ribo-Zero rRNA Magnetic Kit (Epicentre) including the optional RiboGuard RNase inhibitor according to manufacturer's protocol. The concentration was normalised so that each sample contained the same amount of RNA. To 1/3 of the sample 1/10 of the recommended amount of spike-in (ERCC RNA spike-in mix, Ambion) was added, ethanol precipitated, and resuspended in 'Elute, fragment, finish mix' (Illumina). The remaining 2/3 of the sample was ethanol precipitated and resuspended in 15 µl nuclease free water. The sample was heated to 70˚C for 1 min and incubated on ice for 2 min. 5 µl RNase R mixture (Epicentre) was added to the sample before incubation at 37˚C for 30 min. RNase R was removed by phenol/chloroform extraction. The RNA was resuspended in 'Elute, fragment, finish mix' (Illumina). Sequencing libraries were prepared using Truseq stranded RNA LT kit (Illumina) from both Ribo-Zero and Ribo-Zero/RNase R samples, by fragmentation, 1st and 2nd strand cDNA synthesis, 3'-end adenylation, ligation of adaptors, and enrichment of DNA fragments using the manufacturer's protocol The samples were sequenced using the Illumina Illumina HiSeq 2500 platform with 100 bp paired-end reads (AROS Applied Biotechnology)