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| Status |
Public on Feb 18, 2019 |
| Title |
H3K4me3_ChIPSeq_dKO_rep2 |
| Sample type |
SRA |
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| Source name |
H3K4me3_ChIPSeq_dKO
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| Organism |
Mus musculus |
| Characteristics |
strain background: mixed
genotype/variation: Lats1/2;Nestin-Cre dKO
Stage: E12.5
tissue: brain
chip antibody: H3K4me3 antibody (Millipore 07-473)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Each E12.5 mouse brain was dissociated in 1 ml of cold PBS by pipetting. Protein−DNA complexes were crosslinked by incubating in 1% formaldehyde/PBS (Sigma F1635) for 10 min at RT with gentle rotation. Crosslinking was stopped with 125 mM glycine/PBS for 5 min and washed 3 times with cold PBS. Cells were lysed in 1 ml lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100) supplemented with protease and RNAse inhibitors for 10 min at 4ºC with gentle rotation. Nuclei were pelleted at 1800 rcf for 2 min, washed with 1 ml wash buffer (10 mM Tris-HCl [pH 8.0], 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) supplemented with protease and RNAse inhibitors for 10 min, and lysed in 900 µl nuclear lysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-Lauroylsarcosine) for 10 min. Lysed nuclei (300 µl/1.5 ml sonication tube) were sonicated for 5 cycles of 15-sec on and 45-sec off in a Qsonica Q800R2 sonicator. Chromatin was quantitated and diluted with nuclear lysis buffer to ~500 ng/µl and further sonicated for 10 cycles of 15-sec on and 45-sec off to obtain an average DNA fragment size of ~300 bp. Sonicated chromatin was centrifuged at 14,000 rpm for 10 min and the supernatant chromatin was either used for ChIP or flash frozen and stored at −80ºC.
Sequencing libraries were prepared from 10 ng of DNA using the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina with NEBNext Q5 Hot Start HiFi PCR Master Mix according to the manufacturer’s instructions (New England Biolabs) with the following modifications: a second 1:1 Ampure cleanup was added after adaptor ligation and the Ampure size selection step prior to PCR eliminated.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
SJMMNORM054425_G1
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| Data processing |
ChIP-Seq reads were aligned to mm9 (MGSCv37) using the BWA software (version 0.7.12-r1039, default parameter).
Duplicated reads were marked using the Picard software (version 2.6.0-SNAPSHOT) and only non-duplicated reads used for analysis using SAMtools (parameter “-q 1 -F 1024” version 1.2).
Data quality control (QC) follows ENCODE criteria. Relative strand correlation (RSC) values were calculated and the fragment size estimated under the support of R (version 2.14.0) by SPP (version 1.1) with packages caTools (version 1.17) and bitops (version 1.0-6). At least 10 million unique reads with RSC > 1 was required to pass QC. 2 control and 2 Lats1/2 dKO samples passed QC. Bigwig files were generated by extending each read to the best fragment size (the smallest fragment size estimated by SPP) and manually inspected using IGV genome browser for clear peaks and low background noise.
MACS2 (version 2.1.1.20160309) was used to call peaks using the best fragment size with a cutoff of FDR < 0.05
Genome_build: mm9
Supplementary_files_format_and_content: bigwig files were generated using bedtools genomeCoverageBed and bedGraphToBigWig sequentially, bed files were generateted with MACS2 and contain narrow peak calls
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| Submission date |
Sep 16, 2018 |
| Last update date |
Feb 19, 2019 |
| Contact name |
David Finkelstein |
| E-mail(s) |
[email protected]
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| Phone |
9014953931
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| Organization name |
St Jude Children's Research Hospital
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| Department |
Computational Biology
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| Street address |
332 N. Lauderdale St.
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| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38105 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE120014 |
The Hippo Pathway Prevents YAP/TAZ–Driven Hypertranscription and Controls Neural Progenitor Number [ChIP-seq] |
| GSE120016 |
The Hippo Pathway Prevents YAP/TAZ–Driven Hypertranscription and Controls Neural Progenitor Number |
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| Relations |
| BioSample |
SAMN10076271 |
| SRA |
SRX4701624 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3391711_H3K4me3-AB3-SJMMNORM054425_G1-RPK_dkoLNSbrain-12-75.npk.bed.gz |
221.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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