Computational protocol: A context-specific cardiac β-catenin and GATA4 interaction influences TCF7L2 occupancy and remodels chromatin driving disease progression in the adult heart

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Protocol publication

[…] RNA-seq was performed at the Transcriptome and Genome Analysis Laboratory, University Medical Center, Goettingen, in biological triplicates. RNA was extracted, quality and integrity was assessed by Bioanalyzer (Agilent). Libraries were prepared and cDNA libraries were amplified and the size range of final cDNA libraries was determined by applying the DNA 1000 chip on the Bioanalyzer 2100 from Agilent (280 bp). cDNA libraries were sequenced using cBot and HiSeq2000 Illumina (SR; 1 × 50 bp; 51 cycles with single indexing; 6GB ca. 30–35 million reads per sample). Sequence reads were aligned to the mouse reference assembly (UCSC version mm9) using Bowtie 2.0.() For each gene, the number of mapped reads was counted and DESeq2 was used to analyze the differential expression () Gene ontology (GO) analyses were performed using default parameters and stringency in ‘ClueGO’: a Cytoscape plug-in.() The significant ‘GO Biological Processes’ were shown with P ≤ 0.05. [...] TCF7L2 and H3K27ac ChIPs in murine adult cardiac ventricular tissue were performed by 20 min crosslinking with 1.3% formaldehyde and sonicating for 45 cycles. Inputs were pre-cleared for 45 min at 4°C using protein-A-sepharose beads. For immunoprecipitation, 2 μg of anti-TCF7L2, anti-IgG (17–10109, Millipore), anti-GATA4 (sc-25310 X, SantaCruz) or anti-H3K27ac (C15410196, Diagenode) was added to the nuclear extracts and incubated O/N at 4°C. Antibodies were pulled down using protein-A-sepharose beads followed by washing and DNA extraction. For protein complex isolation, proteins were extracted from sepharose beads and supernatants were subjected to immunoblotting. ChIP-seq library preparation was performed using NEBNext Ultra DNA library prep kit for Illumina (E7370) as per manual's instructions. DNA libraries were amplified and sequenced by using the cBot and HiSeq2500 from Illumina (25–30 million reads per sample). Sequence reads were aligned to the mouse reference assembly (UCSC version mm9) using Bowtie2 (). Peak calling was performed with Model Based Analysis of ChIPseq (MACS2) version (). Genes proximal to the bound chromatin regions were identified by GREAT analyses (). Significant ‘GO Biological Processes’ were shown with P ≤ 0.05. Published/public ChIP-seq datasets were used from the following sources: TCF7L2 liver: GSE32513; GATA4, NKX2–5 and TBX3: GSM862697-(33); DNAse-seq: GSM1014166; H3K4me1: GSM769025; RNAPII: GSM918723; H3K27me3: GSM1260017; KLF15: GSM1901940 and CTCF: GSM918756. […]

Pipeline specifications

Software tools Bowtie2, MACS
Application ChIP-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Heart Diseases, Heart Failure