Similar protocols

Pipeline publication

[…] ents were run on 2–3 lanes to obtain an adequate number of reads (Additional file : Table S1C). Matching input DNA was sequenced to obtain a reference for each cell line. The total number of short (35 bp) reads generated per lane varied from 4,509,035 to 27,577,446 with a median of 19,279,525. Raw reads were combined for each histone modification mark and RNA Pol II as well as for input DNA for each of the cell lines. No significant difference was observed in the number of reads generated in the two cell lines for the different histone modification marks or for RNA Pol II (Student’s t-test, P-value range: 0.17-0.83). Alignment to the human genome assembly (NCBI build 37) was performed using Bowtie, allowing for up to 2 mismatches and only one (best) alignment []. The SICER software [] was used to identify qualified peaks (islands) of histone and Pol II binding by comparing sequence reads from immunoprecipitated and input DNA. A consolidating window size of 200 bps was used, a gap size of 200 bps (H3K4me1, H3K4me3 and Pol II) or 600 bps (H3K27me3), effective genome size of 81%, ratio of enrichment between experimental data (PANC-1) and control (hTERT-HPNE) ≥3 and an FDR <0.05. Differentially enriched epigenetic marks across the two cell lines were identified by the SICER-df function. A cis-regulatory element annotation system (CEAS) was used to attain summary statistics on ChIP enrichment peaks based on location in promoters, gene bodies or nongenic regions using the RefSeq database []., Genes were divided into quartiles based on digital expression levels (RPKM values) for each cell line. Global profiling curves were generated for genes in each of the quartile groups by plotting the read distributions (tags were binned into 25 bps bins and trimmed based on Poisson distribution) of different histone modification marks and RNA Pol II within 5,000 bp up- and downstream of transcription start sites (TSS) for RefSeq genes., To assess if a bias existed in read mapping for ChIP-Seq because of chromosomal copy number gain […]

Pipeline specifications

Software tools Bowtie, SICER, CEAS