Computational protocol: Cell-Cycle Control of Bivalent Epigenetic Domains Regulates the Exit from Pluripotency

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Protocol publication

[…] Human Fucci ESCs were sorted into early G1 (DN, double negative), late G1 (KO2, Kusabira orange-2), S phase (AzL, Azami green low), and G2/M (AzH, Azami green high) fractions by FACS on a MoFlo instrument (Beckman Coulter). WA09 hESCs expressing no fluorescence, Cdt1-KO2 only, and Geminin-Az1 only, were used as FACS gating controls. Following FACS isolation, cells were fixed in 1% formaldehyde and quenched with 2.5 M glycine. Approximately 10 million cells per sample were sonicated with a Covaris S220 for 8 min at 200 cycles per burst. Chromatin was collected and then enriched by ChIP with H3K4me3, H3K27me3, H3K27ac, or H3K4me1 antibodies using Protein G Dynabeads (Invitrogen). Sequencing samples were prepared according to the manufacturer’s protocol and sequenced with an Illumina HiSeq instrument (Hudson Alpha). In addition to the ChIP-seq samples of histone marks, input samples were used as sequencing controls. For each sample, ∼40 M reads were obtained by single-end (50 bp) sequencing. Alignment of sequencing reads against human genome (hg19) was performed using the Burrows-Wheeler Aligner (BWA) tool () with the default parameters documented in the bwa-0.5.9 version. Uniquely mapped reads were chosen for the sequencing 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. […]

Pipeline specifications

Software tools BWA, Bowtie, HPeak, ChromHMM
Application ChIP-seq analysis
Organisms Homo sapiens