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Sample GSM1489724 Query DataSets for GSM1489724
Status Public on Mar 06, 2015
Title Mm_e17O_INPUT_rep2
Sample type SRA
 
Source name Mouse e17.5 Embryonic Occipital Cortex
Organism Mus musculus
Characteristics strain: C57BL6J
developmental stage: Embryonic Day e17.5
tissue: Embryonic Occipital Cortex
experiment type: ChIP-seq
chip antibody: N/A
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.
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.
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
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
 
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)
 
Submission date Aug 27, 2014
Last update date May 15, 2019
Contact name Steven K Reilly
E-mail(s) steven.reilly@yale.edu
Organization name Yale University
Department Genetics
Lab Noonan
Street address 333 Cedar St
City New Haven
State/province CT
ZIP/Postal code 06511
Country USA
 
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
Relations
BioSample SAMN03010250
SRA SRX688808

Supplementary file Size Download File type/resource
GSM1489724_Mm_e17O_INPUT_rep2.bw 4.9 Gb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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