Transcriptome Analysis of DNMT and TET Knockouts

Total RNA was extracted and DNAse-I treated using a spin column-based RNA purification kit (Macherey–Nagel). Reverse transcription was performed with 500 ng of RNA using random primers and SuperScriptII (Invitrogen). Primers were designed using Primer 3 [84 (link)] and used for SYBER Green qPCR (Applied Biosystems). All primers sequences are listed in Additional file 9: Table S1. For mRNA sequencing, 100-bp RNA-seq libraries were prepared using 200 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina). Three replicates from independent experiments for each sample for wild-type and DNMT TKO cells and two replicates for TET TKO cells were selected for high-throughput sequencing. Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on an Illumina HiSeq 2500 (Illumina).