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Protocol publication

[…] <5 kb. Plotted is the frequency in 500 bp bins. In case of G9a ChIP-chip data the average log2 (G9a/input) signal was computed in 500 bp bins and subject to filtering using moving average over 6 bins before plotting. Deep sequencing of PolIIS2ph ChIP and input from BIX RH1 treated and untreated HeLa cells has been performed by the EMBL Genomics Core Facility in Heidelberg, Germany. The two ChIP and two input samples have been multiplexed, using NEBNext ChIP-Seq master-mix kit to prepare the libraries. The samples have been sequenced on a 50 bp single-end run on 3 lanes using the Illumina HiSeq 2000 platform. Alignment of the sequenced tags to the hg19 human genome was performed using the CASAVA pipeline 1.8.2, ELAND parameters were: unique matches, 32 base seed, 2 mismatches allowed. This yielded a total of 115.813.632 reads uniquely aligned to hg19 for the untreated IP sample, 126.051.048 for the BIX RH1 treated sample, 127.749.851 for the untreated input and 113.690.799 for the BIX RH1 treated input., Peak calling has been performed on the IP samples vs. their input controls using MACS2 with the parameters: −q0.05−−nomodel−−shiftsize100., This procedure delineated 2046 PolIIS2ph peaks (enriched regions) for the untreated sample and 7712 peaks for the BIX RH1 treated sample. We noted that the BIX RH1 treatment resulted in a higher overall PolIIS2ph enrichment, presumably reflecting a globally more open chromatin environment following the treatment. Therefore, to avoid potential bias we chose to base our analysis on pausing indices relative to gene body signal (see below)., In order to obtain the PolIIS2ph enrichment profiles over the TSS and PAS (), the distance of the PolIIS2ph peaks to the nearest TSS (or PAS) was computed retaining only distances <10 kb away from […]

Pipeline specifications

Software tools BCL2FASTQ Conversion Software, ELAND, MACS
Organisms Homo sapiens