Total RNA from two independent mouse ESC dishes was purified with TRIzol reagent (Life Technologies). In order to remove DNA contaminants, the samples were treated with 1U DNaseI (Thermo Scientific) for 1h at 37°C and the RNA samples were re-purified using TRIzol. The quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The TruSeq RNA Sample Prep Kit v2 (Illumina, Inc, California, USA) was used in the succeeding steps. Briefly, total RNA samples (100-1000 ng) were poly A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples was fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the index for multiplexing Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Caliper GX LabChip® GX (Caliper Life Sciences, Inc., USA). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit HS4000 (Illumina, Inc, California, USA) was used for cluster generation using 10 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the NovaSeq 6000 single end 100 bp using the TruSeq SBS Kit HS4000 (Illumina, Inc, California, USA).