RNA-seq protocol for mESC clones

mESC clones were grown in 24-well plates. Total RNA was extracted from confluent wells using RNeasy Mini Kit (QIAGEN). Libraries for RNA-seq were prepared from 500 ng total RNA using the QuantSeq 3' mRNA-Seq kit (Lexogen) according to manufacturer's instructions. An exception to the instruction was the application of 13 instead of the recommended 12 PCR cycles for library amplification. Libraries were pooled in equal concentrations. Prior to sequencing, a T-fill reaction was performed on a cBot as described previously33 (link), providing the T-fill solution in a primer tube strip. Finally, sequencing was carried out using an Illumina Hiseq-2500 using 50 bp single read v3 chemistry. Raw sequencing data is available from ENA under accession number ERP014134.