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| Status |
Public on Sep 17, 2019 |
| Title |
H3K27me3_ChIPSeq_resected_rep2 |
| Sample type |
SRA |
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| Source name |
Cardiac ventricle tissue
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| Organism |
Danio rerio |
| Characteristics |
tissue: heart ventricle
cell type: enriched cardiomyocytes
experimental batch: 5 days post ventricular apex resection
strain: TuAB
chip antibody: H3K27me3 (Cell Signaling C36B11)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell purification protocol: The apical third of ventricles from 1-1.5 year old fish were extracted and dissociated into single cell suspensions using the murine neonatal heart dissociation kit (Miltenyi). Cardiomyocytes were strained on a 10 micrometer nylon mesh while other cell types in the flowthrough were discarded. The strainer with the attached cells was then processed as a cell pellet, including 30 minute fixation in methanol-free formaldehyde (1% final concentration) and washing with sterile PBS containing protease inhibitors. Lysates were clarified from sonicated cells and histone-DNA complexes were isolated with antibody. Libraries were prepared on a BRAVO automated liquid handling platform (Agilent) as previously described (Busby M. et al., 2016).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
ChIP libraries were indexed, pooled, and sequenced on Illumina NextSeq-500 sequencers at the Broad Institute sequencing center. Reads were aligned by the Broad Genomics Platform with BWA (Li and Durbin 2009, v0.7.10) using default parameters against the Danio rerio danRer7 genome assembly ((bwa v. :0.5.9-tpx; bwa aln -q 5 -l 32 -k 2
out1.sai fastq_1 file; bwa aln -q 5 -l 32 -k 2 out2.sai fastq_2 file; bwa sampe -T -P -f
out_sam_paired.sam Danio_rerio.fasta out1.sai out2.sai fastq_1 _fastq_2). H3K4me3-
associated read peaks were called using MACS2 using the following parameters: Macs2
callpeak -t sample.bam -c WCE.bam -f BAM -g 1.5e9 -B -p 1e-3 --to-large --nomodel -
-broad --extsize 147). Quality scores of bam files were adjusted using the CleanSam
utility version 2.18.11, and files were converted to fastq and aligned against the GRCz10
Zebrafish genome assembly using bowtie2 v. 2.3.2 with default parameters, and -p 8.
Mapped reads were marked with PICARD v. 2.8.1 MarkDuplicates function with
parameters MAX_SEQUENCES_FOR_DISK_READ_ENDS_MAP=50000
MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=8000
SORTING_COLLECTION_SIZE_RATIO=0.25
REMOVE_SEQUENCING_DUPLICATES=false TAGGING_POLICY=DontTag
REMOVE_DUPLICATES=false ASSUME_SORTED=false
DUPLICATE_SCORING_STRATEGY=SUM_OF_BASE_QUALITIES
PROGRAM_RECORD_ID=MarkDuplicates
PROGRAM_GROUP_NAME=MarkDuplicates READ_NAME_REGEX=<optimized
capture of last three ':' separated fields as numeric values>
OPTICAL_DUPLICATE_PIXEL_DISTANCE=100 VERBOSITY=INFO QUIET=false
VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5
MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false
GA4GH_CLIENT_SECRETS=client_secrets.json PN:MarkDuplicates . The resulting
bam files were sorted and filtered with samtools to exclude sam flags 1796. Read count tables were generated using bedtools v. 2.26.0 (Quinlan et al. 2010) multicov -p utility versus a .bed file containing promoter regions spanning 2Kb up and downstream of each TSS (based on ENSEMBL release 89). Differential isoform expression was estimated with DESeq2 (Love et al 2014) to obtain log2
fold-change data and significance levels (p-values) between uninjured and 5 dpa
samples. IGVtools utility 'count' was used on aligned bam files to compute the average alignment over 200 base windows across the genome and generate binary tiled data (.tdf) files (Thorvaldsdottir et al. 2013).
Genome_build: danRer10
Supplementary_files_format_and_content: csv files are a DEseq2 estimations of changes in read density across 4-kb windows centered on annotated TSSs (danRer10/ENSEMBL 89) between experimental groups.
Supplementary_files_format_and_content: tdf files are binary tiled data files of aligned ChIP-Seq data from the IGVtools count utility that contain average alignment counts across the genome and can viewed in the IGV genome browser.
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| Submission date |
Mar 22, 2017 |
| Last update date |
Sep 17, 2019 |
| Contact name |
Raz Ben-Yair |
| E-mail(s) |
razbe@weizmann.ac.il
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| Phone |
6177553081
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| Organization name |
MGH
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| Department |
CVRC
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| Lab |
Caroline Burns
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| Street address |
13th st.
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| City |
Charlestown, MA |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
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| Platform ID |
GPL20828 |
| Series (2) |
| GSE96928 |
H3K27me3 deposition over sarcomeric and cytoskeletal promoters is required for cardiomyocyte cytokinesis and wound invasion during zebrafish heart regeneration [ChIP-seq] |
| GSE96930 |
H3K27me3-mediated silencing of structural genes is required for zebrafish heart regeneration |
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| Relations |
| BioSample |
SAMN06628153 |
| SRA |
SRX2661983 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2546205_K27me3amp2.tdf.gz |
182.9 Mb |
(ftp)(http) |
TDF |
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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