Examining DNA Methylation Dynamics in Tet3 Knockout Mouse ESCs

Tet3^+/+, Tet3^m/m and Tet3^–/– mouse ESCs were differentiated to NE (-LIF + RA for 3 days). DNA was extracted and subjected to hMeDIP (Active Motif hMeDIP Kit #55010) following manufacturer guidelines, and amplified using NEBNext HiFi 2x PCR Master mix. Libraries were cleaned with AMPure XP beads (#A63880) and subjected to 75 bp paired-end sequencing using Illumina NextSeq 500 platform at the Einstein Epigenomics Core. Adapters were trimmed using TrimGalore and sequencing reads were mapped to the mouse genome (mm10) using bowtie2 (v2.4.5)^{50 (link)} with the following parameters: --local --sensitive --very-sensitive-local --no-unal --no-mixed --no-discordant –phred33 -I 10 -X 700. The BAM files were balanced to the sample with the lowest reads using samtools and were used to call peaks using MACS2 (v2.2.7.1)^{51 (link)} with the following parameters: -p 0.0001 -f BAMPE --keep-dup all. BED files with genomic coordinates were annotated using ChIPseeker (v1.36.0). IgG bigwig was subtracted from Tet3^+/+, Tet3^m/m and Tet3^–/– bigwigs using function bigwigCompare --operation subtract from deepTools. The output bigwig files were used to make line plots using function plotProfile from deepTools.