|Public on Apr 28, 2016
|MCF7 cell line
|cell line: MCF7
cell type: breast cancer cells
|For H2A.Z knockdown experiments, 10umol/l scramble and H2A.Z siRNA were used to transfect MCF7.
|MCF7 was cultured in DMEM with 10% FBS
|MCF7 were crosslinked with 1% formaldehyde for 10min and then incubated with nuclear extraction buffer (20mM Hepes-KOH ph7.5, 10mM KCl, 1mM EDTA, 0.2% NP40, 1mM DTT and 10% glycerol) to isolate nuclei. The nulei in sonication buffer (20mM Tris-HCl pH8, 150mM NaCl, 2mM EDTA, 0.1% SDS, 1% TritonX-100) was sheared in Misonix Sonicator. The further chromatin immunoprecipitation was implimented with RNAP2 antibody (05-623 Millipore) .
The library construction followed the protocol in kit of NEBNext® ChIP-Seq Library Prep Reagent Set (E6200, New England Biolab) with the modfication that single index primer in place of dual primers was used in the final step of PCR amplication.
The libraries were indexed with Illumina barcode. Five indexed libraries were finally pooled into one lane and sequenced in Hiseq2500.
|Illumina HiSeq 2500
|Cross-linked, sonicated genomic input DNA
|First the reads with aligned to hg19 using bowtie2 with -M 1 option.
Aligned reads were processed and filtered using the spp R package to filter out low quality reads.
ChIP-seq and input reads were mapped to RefSeq annotated genes calculating the background-subtracted RNA Polymerase II density at the TSS and gene body of each gene
Supplementary_files_format_and_content: Comma-separated files (CSV) of each RefSeq isoform with library-size and length normalized read density of ChIP or Input reads mapping to the TSS or gene body of each annotated isoform
|Aug 28, 2014
|Last update date
|May 15, 2019
|Peter J Park
|Harvard Medical School
|Center for Biomedical Informatics
|10 Shattuck St
|The functional impact of RNA Polymerase II pausing across multiple mamamlian cell types