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Pipeline publication

[…] encing analysis. To eliminate noise and to take into account the unequal total numbers of reads for different sample, we identified the ChIP signal enriched regions: the 2.5 kb region from 1 kb upstream of the TSS to 1.5 kb downstream of the TSS as H3K4me3 signal-enriched region. For each bivalent gene obtained from , we calculated the normalized RPM (reads per million) at ChIP signal enriched regions for H3K4me3 and H3K27me3, respectively. A list of bivalent genes was compiled that show a >2-fold RPM change between any of the adjacent cell-cycle time points and included these in heatmap analysis. For ChIP-seq with immunoprecipitations against H3K27ac and H3K4me1, reads were mapped using Bowtie (). HPeak () was used to call peaks from mapped reads. Gene Ontology (GO) analysis was conducted using R ( Differential binding sites were identified using a conditional binomial model based on counts falling in specific peak regions of each sample. Histone tracks in H1 hESCs (H3K4me1 and H3K27ac) from ENCODE and chromatin state dynamics (ChromHMM) tracks () were downloaded for inclusion in ChIP-seq analysis. The categories considered as strong or weak enhancers had overlapping H3K27ac peaks. qChIP was performed as previously described using the Kapa SYBR Fast Kit (KK4602, KAPA Biosystems) according to the manufacturer’s instructions on a ViiA7 Real-Time PCR instrument (Life Technologies). Antibodies for ChIP were as follows: H3K4me3 (ab8580 and ab1012, Abcam), H3K27me3 (ab6002, Abcam), H3K4me1 (ab8895, Abcam), H3K27ac (ab4729, Abcam), MENIN (ab2605, Abcam), Wdr5 (ab56919, Abcam), MLL2/TRX2 (A300-113A, Bethyl Laboratories), and Cdk2 D12 antibody (sc-6248. Santa Cruz Biotechnology). See for primer sequences., For genome editi […]

Pipeline specifications

Software tools Bowtie, HPeak, ChromHMM