Computational protocol: Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells

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Protocol publication

[…] Reads were aligned on the UCSC Homo sapiens hg19 genome using TopHat2 (). Differential expression analysis was performed as described in (), using the Bioconductor package edgeR (). Only genes with at least 1 read per million (RPM) in at least two of the samples were kept for subsequent analyses. Enriched Gene Ontology terms and KEGG pathway were identified using Gene Set Enrichment Analysis () with genes pre-ranked according to their fold change induced by siMBD2 treatment. [...] Reads were aligned on the UCSC Homo sapiens hg19 genome using Bowtie (). Duplicate reads were filtered using SAMTools () to limit PCR induced biases. Preliminary peak detection was performed using Model-based Analysis of ChIP-Seq (MACS) (). Over sequenced regions were defined as 500 bp regions containing more than three times the median number of reads in at least one of the two inputs. Peaks were filtered against these over sequenced regions, as they represent suspicious false positive regions. Peaks in HMEC-hTERT and HMLER were compared using MAnorm (), and peaks common to both cell lines or cell line specific peaks (P value ≤ 0.01) were determined. Read density visualization at specific locations were drawn with Sushi.R (). K-mean clusterization of peaks and subsequent visualization were performed with seqMINER (). Integrative analysis of ChIPseq, RNAseq and ChIP-chip data was performed using the Bioconductor package Repitools (). Top enriched motifs in peaks were obtained using the RSAT oligo-diff tool (). […]

Pipeline specifications

Software tools Bowtie, SAMtools, MAnorm, Sushi.R, seqMINER, Repitools, RSAT
Application ChIP-seq analysis
Organisms Homo sapiens