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| Status |
Public on Jan 28, 2016 |
| Title |
KMT2D ChIP-Exo 2 |
| Sample type |
SRA |
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| Source name |
Cardiomyocytes
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| Organism |
Mus musculus |
| Characteristics |
antibody: KMT2D
strain: C57BL/6
cell type: ES-cell derived cardiomyocytes
developmental stage: NA
replicate: replicate 2
genotype: E14 Tg(Nkx2-5-EmGFP) mouse ES cells
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| Treatment protocol |
Both WT and mutant embryos were harvested at embryonic day E11.5 and embryonic hearts were dissected in cold PBS. Hearts were flash frozen in liquid nitrogen and kept at -80˚C.
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| Growth protocol |
Male mice with a heterozygous Kmt2d floxed allele and expressing the Tnnt2::Cre recombinase (Tnnt2::Cre; Kmt2d fl/+) were crossed to female mice homozygous for Kmt2d floxed alleles and the Rosa26-Cre reporter mTmG (Kmt2d fl/fl; mTmG/mTmG). The females will carry litters with the WT control genotype (Tnnt2::Cre; Kmt2d fl/+; mTmG/+) and the mutant genotype (Tnnt2::Cre; Kmt2d fl/fl; mTmG/+).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-Exo Library Prep: All adapter and primer sequences were obtained from Serandour et al., Genome Biology, 2013. Briefly, immunoprecipitation was performed using a mixture of Protein G magnetic beads, anti-KMT2D antibody, and crosslinked KMT2D-DNA complexes. Beads were collected using a magnet and all supernatant (containing unbound material) was removed. Several enzymatic reactions were then performed while the immunoprecipitated KMT2D-DNA complexes were still bound to the beads; a number of washes preceded each step. First, complexes were treated with T4 DNA polymerase, E. coli Pol I large (Klenow) fragment, and T4 polynucleotide kinase to ensure all dsDNA fragments were blunt-ended with 5' phosphates. Next, P7 adapter was ligated to each fragment using T4 DNA ligase. Following ligation, nicks were repaired using Phi29 polymerase. KMT2D-DNA complexes were then digested with Lambda exonuclease, which acts only in the 5' to 3' direction. This step leaves behind single-stranded DNA except where crosslinked protein protects the DNA from digestion. Following this first digestion, a second digestion was performed using RecJf, a fusion protein of RecJ and maltose binding protein. This enzyme specifically digests single-stranded DNA in the 5' to 3' direction. Finally, immunoprecipitated KMT2D-DNA complexes were eluted and crosslinks were reversed overnight (65C, 0.54M NaCl). DNA was isolated from each sample. Complimentary strand synthesis was completed by denaturing the DNA and then treating with Phi29 polymerase following the addition of a primer complimentary to the P7 adapter sequence. DNA was isolated and P5 adapter was ligated to each strand using T4 DNA ligase. DNA was again isolated and amplified using a pair of universal and barcoded primers. Amplified fragments between 200 and 400bp were isolated from an agarose gel and purified. This DNA was then clustered on an Illumina flow cell and sequenced on an Illumina HiSeq 4000.
Both WT and mutant hearts were thawed on ice and pooled into 3 replicates of 30 hearts each. Samples were crosslinked with 1% formaldehyde, lysed with cell lysis and nuclei lysis buffer, and sonicated to shear chromatin into 200-600 bp fragments. Each replicate was further split into input and 3 samples. Chromatin immunoprecipitation was performed with 3 ug of anti-H3K4me1/2/3 antibody (Abcam) overnight with Protein G Dynabead (Life Technologies). After washing, DNA was eluted, reverse-crosslinked and precipitated with AmPure XP magnetic beads (Beckman Coulter).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Differentiated using a protocol from Wamstad et al., Cell, 2012.
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| Data processing |
Sequencing reads were de-multiplexed using CASAVA version 1.8.2.
Reads are trimmed using the fastq-mcf program (https://code.google.com/p/ea-utils/).
The filtered reads are analyzed using the Fastqc program (ttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
The reads are aligned to the genome assembly of choice using bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2).
Samtools is used to keep only the reads that do not map uniquely to the genome (mapq score >= 30) (http://samtools.sourceforge.net/).
bam2bed is used to convert bam files into bed files, the genome-wide shift between + and - strand tags is calculated in a manner adapted from the Kundaje method (Neph et al, Nature, 2012; Landt et al., Genome Res, 2012; http://bedops.readthedocs.org).
The genome is divided into 20bp bins. The tag density is defined as the number of tags that map to within 75bp of each genomic bin (Landt et al., Genome Res, 2012).
If input chromatin is provided, then the tag density of input is calculated identically. Tag densities are normalized as follows: tagDensity = #binsInGenome * ((#tags/#totalTags) - (#inputTags/#totalInputTags))
If input chromatin is not provided, then the tag densities are normalized as follows: tagDensityOfGenomicBin = #tagsAtBin(step06.1) * #binsInEntireGenome / #totalTagsInDataset
Bins representing either 6-fold enrichment over input (tag density ≥ 30) for H3K4me1/2/3 or roughly 6.64-fold over background (tag density ≥ 100) for KMT2D are accepted as peaks.
Enriched bins ("peaks") are merged together throughout the genome, merged bins representing signal peaks spanning multiple genomic bins are retained.
In order to compare peak scores and perform differential enrichment, peaks are annotated with total tag density within the peak region (Paige et al, Cell, 2012).
Genome_build: mm9
Supplementary_files_format_and_content: BigWig files contain tag density data displayable as a continuous graph. BigBed files contain genomic intervals defined as "peaks" (see manuscript) stored in an indexed binary format; plain bed files contain the same but in plain text format.
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| Submission date |
Nov 18, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Siang-Yun Ang |
| E-mail(s) |
siangyun.ang@gmail.com
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| Organization name |
Gladstone Institute of Cardiovascular Disease
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| Lab |
Benoit Bruneau
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| Street address |
1650 Owens St
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE75151 |
KMT2D regulates specific programs in heart development via histone H3 lysine 4 dimethylation |
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| Relations |
| BioSample |
SAMN04276941 |
| SRA |
SRX1437360 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1944104_MLL2_KMT2D_Exo_conc03.1_mm9.chr_peaks.bb |
299.0 Kb |
(ftp)(http) |
BB |
| GSM1944104_MLL2_KMT2D_Exo_conc03.1_mm9.chr_tagDensity.bw |
515.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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