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| Status |
Public on Mar 06, 2015 |
| Title |
Mm_e17F_H3K27ac_rep1 |
| Sample type |
SRA |
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| Source name |
Mouse e17.5 Embryonic Frontal Cortex
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6J
developmental stage: Embryonic Day e17.5
tissue: Embryonic Frontal Cortex
experiment type: ChIP-seq
chip antibody: H3K27ac (Abcam ab4729)
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| Treatment protocol |
All tissues were briefly homogenized and crosslinked with 1% formaldehyde at room temperature with rotation for 15 minutes. Crosslinking was quenched with 150 mM glycine. Tissue were harvested by centrifugation at 1500g for 5 min at 4C. Pelleted tissue was washed twice with cold PBS, flash frozen and stored at -80°C.
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| Growth protocol |
Human brain tissue and rhesus brain tissue were thawed and micro dissected. Mouse cortex tissue was directly prepared after harvesting. Rhesus and mouse were harvested from timed pregnant mothers.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei were extracted in six pellet volumes of lysis buffer I (50 mM Tris pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x Roche Complete Protease inhibitor, 5 mM sodium butyrate) and incubating on ice for 15 minutes. Tissue was homogenized with a dounce homogenizer and nuclei were harvested by centrifugation at 2000g for 10 minutes at 4°C. Nuclei were resuspended in five pellet volumes of nuclear lysis buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 0.2% SDS, 1x Roche Complete Protease inhibitor, 5 mM sodium butyrate) and incubated on ice for 20 minutes. 300 mL nuclear lysates in 1.5 mL tubes were sonicated using a Misonix S4000 with 431A cup horn (10 second pulses, 10 second rest, 20 minutes total, 2°C maintained by circulating chiller). Following sonication insoluble material was pelleted by centrifugation at 16000g for 10 minutes at 4°C. A small sample of soluble crosslinked chromatin was purified, checked for correct sonication size range and DNA concentration was measured. For H3K27ac, 10-25 ug of chromatin was diluted to 450 mL with dilution buffer (16.7 mM Tris pH 8, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, 1x Roche Complete Protease inhibitor, 5 mM sodium butyrate) and added to 50 uL of Protein G Dynabeads (Invitrogen) prebound with 2 mg of H3K27ac antibody.For H3K4me2, 10-25 ug of chromatin was diluted to 450 mL with dilution buffer (16.7 mM Tris pH 8, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, 1x Roche Complete Protease inhibitor) and added to 50 uL of Protein G Dynabeads (Invitrogen) prebound with 10 mg of H3K4me2 antibody. Samples were incubated overnight at 4°C with rotation. Beads were precipitated on magnet stand and washed eight times with 1 mL of wash buffer (100 mM Tris pH8.0, 500 mM LiCl, 1% NP-40, 1% deoxycholic acid, 1x Roche Complete protease inhibitor) and once with TE. Immunoprecipiated chromatin was eluted from beads with 50 mL of elution buffer (TE + 1% SDS) at 65°C for 10 minutes. Crosslinks were reversed overnight at 65°C. Chromatin was treated with RNAseA and proteinase K then purified with Qiagen PCR cleanup kit. Concentration of eluted chromatin was measured with PicoGreen (Invitrogen).
Standard Illumina Multiplexed Paired-End Library Prep
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Bowtie (v0.12.9), cd /panfs/sequencers/sequencerP/runs/131130_SN356_0279_BC30AWACXX/Data/Intensities/BaseCalls/Unaligned-Skr2/Project_Skr2/Sample_SR_40_Index5; zcat *.gz > ~/Scratch/Steve/Hu_12Fpcw_INPUT_rep1.fastq.gz; source ~/.bashrc; cd ~/Scratch/Steve/; bowtie -q -n 2 -l 28 -m 1 -p 8 -5 1 -3 5 -t /home/jl56/GENOME/hg19/dna/hg19_nh ./Hu_12Fpcw_INPUT_rep1.fastq.gz ./Hu_12Fpcw_INPUT_rep1.bowtie
Custom perl script , Cotney et al, Cell 2013.
RSEQTools (0.6.0), cat Hu_12Fpcw_INPUT_rep1.bowtie | bowtie2mrf genomic > ../mrf/Hu_12Fpcw_INPUT_rep1.mrf
RSEQTools (0.6.0) and wigToBigWig, cat Hu_12Fpcw_INPUT_rep1.mrf | mrf2wig Hu_12Fpcw_INPUT_rep1; cat Hu_12Fpcw_INPUT_rep1_chr*.wig | grep -v track > Hu_12Fpcw_INPUT_rep1.wig; wigToBigWig -clip Hu_12Fpcw_INPUT_rep1 /home/jl56/GENOME/hg19/dna/hg19_nh.chrom.sizes Hu_12Fpcw_INPUT_rep1.bw
Genome_build: hg19 for human samples, rheMac2 for rhesus samples, mm9 for mouse samples, Gencode V10 gene annotation
Supplementary_files_format_and_content: bigWig (signal tracks), bed (peak calls), bam (anonymized human reads), fastq (raw rhesus and mouse reads)
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| Submission date |
Aug 27, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Steven K Reilly |
| E-mail(s) |
steven.reilly@yale.edu
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| Organization name |
Yale University
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| Department |
Genetics
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| Lab |
Noonan
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| Street address |
333 Cedar St
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06511 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE60838 |
Evolutionary changes in promoter and enhancer activity during corticogenesis (non-human) |
| GSE63649 |
Evolutionary changes in promoter and enhancer activity during human corticogenesis |
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| Relations |
| BioSample |
SAMN03010239 |
| SRA |
SRX688797 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1489713_Mm_e17F_H3K27ac_rep1.bw |
676.9 Mb |
(ftp)(http) |
BW |
| GSM1489713_Mm_e17F_H3K27ac_rep1_regions.bed.gz |
631.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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