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|Public on Oct 18, 2016
|ChIP-seq H3K27Me3 D Hhex F
genetic modification: ΔERT2 + Hhex-F
|DNA was fragmented in a Covaris Sonicator (30 minutes, 4°C) then protein-DNA complexes immunoprecipitated from clarified chromatin fractions using H3K27Me3 (2μg; Millipore: 07-449) or H3K4Me3 (2μg; Millipore: 07-473) antibodies and 30μl of Protein A-sepharose. For Hhex-Flag ChIP, 30μl of anti-Flag (M2)-agarose beads (Sigma) was added directly to clarified chromatin fractions. Following reversal of crosslinks and RNaseI and Proteinase K treatments, extracted DNA was purified using the QIAquick purification kit.
ChIP-Seq DNA libraries were constructed using TruSeq Nano DNA sample preparation according to the manufacturer’s instructions.
|Illumina NextSeq 500
|Reads were aligned to the mouse genome by Rsubread
For the H3K4Me3 samples, read counts were summarized in a region 150bp up and down from the transcriptional start site of each gene using the Rsubread featureCounts functions and NCBI annotation.
For the H3K27Me3 samples, read counts were summarized within the gene body of each gene using the Rsubread featureCounts functions and NCBI annotation.
Peak calling was performed on the M2 Flag samples using MACS.
Supplementary_files_format_and_content: Tab-delimited text files were written from the R prompt for the H3K4Me3 and H3K27Me3 read counts; columns represent the Entrez Gene ID, Gene Symbol, total gene length (total base count of all exons) and the number of reads mapping to that gene. Bed files for the M2 Flag peak calls were generated by MACS2. They contain the peak summit locations for each peak; columns represent chromosome name, start position of peak, end position of peak, peak ID, and the pileup height at peak summit (the -log10(pvalue) for the peak summit).
|Oct 14, 2015
|Last update date
|May 15, 2019
|Olivia Newton-John Cancer Research Institute
|145 Studley Road
|Acute myeloid leukemia requires Hhex to enable PRC2-mediated epigenetic repression of Cdkn2a (ChIP-Seq)
|Acute myeloid leukemia requires Hhex to enable PRC2-mediated epigenetic repression of Cdkn2a