RNA-sequencing sample preparation and analysis

Mouse embryonic stem cells were collected with accutase (5 min, 37 °C) and counted. Cells (3 × 10⁵) were lysed with 300 µl TRIzol reagent. RNA was extracted using the Direct-Zol RNA extraction kit from Zymo. Library preparation was performed after Illumina TruSeq Stranded mRNA-seq according to the manufacturer protocol. Reads were mapped to the Mus musculus genome (build mm9) using STAR⁵⁸, using the following options: --outSJfilterReads Unique --outFilterType BySJout --outFilterMultimapNmax 10 --alignSJoverhangMin 6 --alignSJDBoverhangMin 2 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outSAMtype BAM SortedByCoordinate --seedSearchStartLmax 50 --twopassMode basic. Gene expression was quantified using qCount from QuasR package⁵⁹ using the ‘TxDb.Mmusculus.UCSC.mm9.knownGene’ database for gene annotation (Bioconductor package: Carlson M and Maintainer BP. TxDb.Mmusculus.UCSC.mm9.knownGene: Annotation package for TxDb object(s); R package v.3.2.2). Active promoters were defined as genes with log₂[RPKM + 0.1] higher than 1.5.