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| Status |
Public on Jul 15, 2019 |
| Title |
P1_3MI_rep1_H3K27me3 |
| Sample type |
SRA |
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| Source name |
Mouse ventricle below left anterior descending artery ligation plane
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| Organism |
Mus musculus |
| Characteristics |
strain: ICR/CD1
surgery performed at developmental age: Postnatal day 1
sample collected at post-surgical day: day 3
surgery type: MI
antibody: H3K27me3
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Each heart was dissected in ice-cold PBS and a transverse cut on ligation plane was made, separating it into above ligation plane (ALP) part and below ligation plane (BLP) part. BLP parts were flash-frozen with liquid nitrogen and stored in -80°C for RNA-Seq. The collected tissue samples were ground to powder in liquid nitrogen, and crosslinked with 2% formaldehyde in PBS for 30 min and quenched with 0.125M glycine for 10 min at room temperature. Crosslinked samples were then washed with cold PBS and further homogenized with a Dounce tissue grinder. Samples were collected by a brief spin, and treated with 10 mM Tris-HCl (pH 8.0), 10mM NaCl and 0.2% NP-40 for 30 min to collect nuclei. After nuclear extraction, chromatin was sheared on a Bioruptor Pico (Diagenode) for 20 cycles (30 sec on/ 30 sec off for each cycle) at 4°C in sonication buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.25% sarkosyl, 1 mM DTT, 1x cOmplete Protease Inhibitor Cocktail (Roche), and 1 mM PMSF, pH 8.0). After sonication, 1% of the sonicated chromatin from each sample was taken out as “input” samples. The remaining sonicated chromatin was evenly split for H3K27ac or H3K27me3 ChIP. Sonication buffer was added to make the final volume to 1 mL. NaCl was then added to a final concentration of 300 mM for each sample. 1 µg H3K27ac antibody (Diagenode, C15410196) or H3K27me3 (Diagenode, C15410195) was added to each sample and incubated at 4°C overnight with gentle rotation. The next day, 30 µL of pre-washed Dynabeads Protein G (Invitrogen, 10004D) was added to each sample for a two-hour incubation. After that, the beads were washed twice with 1 mL RIPA 0 buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, pH 8.0), twice with 1 mL RIPA 0.3 buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, 300 mM NaCl, pH 8.0), twice with 1 mL LiCl wash buffer (250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), and finally twice with 1 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). For each ChIP sample or input sample, 100 uL of SDS elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) was added and incubation was done at 65°C overnight on a ThermoMixer (Eppendorf) at 1000 rpm. The next day, supernatant was collected and further treated with 0.5 µg RNaseA (Sigma, 11119915001) for 30 min at 37°C, followed by 20 µg Proteinase K (NEB, P8107S) treatment at 37°C for 2h. DNA was recovered using MinElute PCR Purification Kit (QIAGEN, 28004) according to manufacturer’s protocol.
ChIP-Seq libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) according to manufacturer’s protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
P1_3MI_H3K27me3.fc.signal.bw
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| Data processing |
Quality control of RNA-Seq data was performed using FastQC Tool (Version 0.11.4).
ChIP-seq raw reads were aligned to mouse (mm10) genome assembly using Bowtie2 (Version 2.3.4) with default parameters.
For H3K27ac samples, duplicated reads were removed from further analysis. MACS2 (Version 2.1.1) was first applied to each sample to perform peaking calling using the “-nomodel” parameter and was next applied to merged pseudo samples of the biological duplicates14. Only the peaks that are present in both replicates as well as the merged pseudo sample, defined by at least 50% overlapping of the peak coordinates, were kept as real peaks for further analyses.
To compare ChIP-seq signal intensities between MI and sham samples, diffbind (Version 1.16.3) was applied to calculate the normalized ChIP-seq enrichment score with summits=250. Cutoff values of absolute fold change greater than 2.0 and false discovery rate less than 0.05 were used for differential peak analysis between sample groups.
The normalized peak scores calculated using the TMM function in edgeR were plotted in the heatmaps to show peak intensity across timepoints.
For H3K27me3 samples, duplicates and X-chromosome mapped reads were removed. MACS2 was applied to each biological sample using “--broad-cutoff 0.01 –nomodel” parameter.
Diffbind was applied to calculate the differentially bound peak with summits=500. Fold change value of two and FDR less than 0.05 were used as cutoffs.
Genome_build: mm10
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| Submission date |
Dec 14, 2018 |
| Last update date |
Jul 15, 2019 |
| Contact name |
Zhaoning Wang |
| E-mail(s) |
zhw063@health.ucsd.edu
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| Organization name |
UC San Diego
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| Department |
Cellular and Molecular Medicine
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| Lab |
Bing Ren Lab
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| Street address |
9500 Gilman Drive
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE123867 |
Epigenome profiling of neonatal heart regeneration |
| GSE123868 |
Mechanistic basis of neonatal heart regeneration revealed by transcriptome and histone modification profiling |
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| Relations |
| BioSample |
SAMN10594250 |
| SRA |
SRX5131401 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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