Raw sequencing reads were first estimated, and no preprocessing was performed because of high reads quality. Then, reads were mapped to human genome GRCH38 using BWA (0.7.15-r1140) with default parameters. Low-quality mapped reads and PCR duplicates were removed using SAMtools (v1.2) and PICARD (1.138) (http://broadinstitute.github.io/picard). Promoters were defined as upstream 1 kb and downstream 0.5 kb of TSS. Meta gene profile and TSS profile were generated using deepTools2 (3.0.1). Normalized reads distribution profile were produced using deepTools2 and visualized using the University of California, Santa Cruz (UCSC) genome browser. MACS2 was used to identify enrichment peaks for Bach1, EZH2 (with default parameters for “sharp” peaks), and H3K27me3 (with default parameters for “broad” peaks). The accession number for the RNA-seq and ChIP-seq data of WT and Bach1-KO hESCs reported in this paper is GEO: GSE113817. The accession numbers for published ChIP-seq data of Bach1, EZH2, and H3K27me3 are GEO: GSE31477, GEO: GSE29611, and GEO: GSM466734, respectively.

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