Experiment name : give a name for the MACS run (siNT_ESR1_MACS).Select the tools MACS14 in the Mardi 19 ChIP-seq section and fill the form as below Peak calling will be performed using MACS (version 1.4.1). and an input sample (only one replicate is available, MCF-7_input_r3_.*.bam).Note that r1, r2 and r3 stand for replicate 1, 2 and 3. a ChIP sample ( e.g siNT_ER_E2_r1_.*.bam).In the next section, we will search for ER (Estrogen Receptor) binding sites in control samples (ChIP Estrogen Receptor on MCF-7 cell line treated with E2). Thus, in order to illustrate the peak-calling procedure, BAM files have been split into several files each of them containing the reads aligned to a given chromosome. Cloning shared data.Īnalysis of the whole dataset can be time consuming. Goal : Retrieve BAM files that contain alignment results. How many reads were produced (# of Spots) ?.Is this experiment single-end or paire-end sequencing ?.What is the HTS platform used to sequence this sample ?.Both web sites propose search engines to query their databases. Raw data are available from the Sequence Read Archive ( SRA) database (including 454, IonTorrent, Illumina, SOLiD, Helicos and Complete Genomics). For HTS data, GEO provides both processed data (such as *.bam, *.bed, *.wig files) and links to raw data. Data may be related to array- or sequence-based technologies. Gene Expression Omnibus ( GEO) is a public repository that provide tools to submit, access and mine functional genomics data. Step 1 : Find datasets on Gene Expression Omnibus Together, these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility, and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer. Chromatin loops at the TFF locus involving ESR1-bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen-ESR1-mediated interactions between cis-regulatory elements. The GATA3-mediated redistributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. These global genomic changes altered the ESR1-binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3-mediated redistribution of ESR1 binding. GATA3 silencing resulted in a global redistribution of cofactors and active histone marks prior to estrogen stimulation. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. GATA3 is an ESR1-cooperating transcription factor mutated in breast tumors however, its genomic properties are not fully defined. Differences in enhancer occupancy by ESR1 contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. AbstractĮstrogen receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. The corresponding abstract of this article is provided below. Importantly, they demonstrated that knock-down of GATA3 through siRNA strongly affect ESR1 binding sites. The authors used ChIP-Seq technology in order to systematically identify ESR1 binding regions across the human genome. Goal: how do we obtain the datasets referenced in a publication ?įor this tutorial we will use CHiP-Seq datasets produced recently by Theodorou et al. Step 6 : peak-calling using MACS on ER ChiP-Seq after siGATA3 treatment.Step 5 : Visualizing the results with Integrated Genome Browser (IGV).Step 4 : peak-calling using MACS on ER ChiP-Seq.Step 1 : Find datasets on Gene Expression Omnibus.Analysis of ChIP-seq data ChiP-Seq: Peak calling using MACS Contents
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