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Sample GSM1036117

Query DataSets for GSM1036117

Status

Public on May 22, 2013

Title

Input_H3K27ac_H3K4me2_e14.5cortex

Sample type

SRA

Source name

Embryonic cortex

Organism

Mus musculus

Characteristics

embryonic day: E14.5
strain: C57BL/6
tissue: Embryonic cortex

Treatment protocol

Pregnant mothers were euthanized and embryos were removed and placed in cold PBS. Tissue (forelimbs, hindlimbs, cortex) was dissected with forcepts.

Growth protocol

Normal mouse gestation to day E10.5, E11.5, or E14.5

Extracted molecule

genomic DNA

Extraction protocol

Total RNA was extracted with RNEasy kit (Qiagen) for RNA-seq. For each ChIP-Seq and ChIA-PET, limb bud tissue was crosslinked with 1% formaldehyde at room temperature and stored at -80°C. Chromatin was extracted and sheared by sonication. Soluble chromatin was combined Protein G Dynabeads prebound with appropriate antibodies at 4C overnight. Beads were collected with magnet and washed 5 or 8x. For ChIP-seq, chromatin was eluted with TE+1%SDS at 65C for 10 minutes. Crosslinks were reversed at 65C overnight then chromatin was purified with PCR cleanup kit (Qiagen). For ChIA-PET, chromatin was end-repaired, ligated to ChIA-PET adaptors, and the 5’ ends of the adaptors was phosphorylated. Chromatin was eluted as described above, followed by ligation of chromatin complexes and reversal of crosslinking (65C overnight). DNA was purified with phenol:chloroform extraction and ethanol precipitation.
ChIP-seq libraries were constructed with the standard procedure included with the Illumina ChIP-Seq kit but paired-end adaptors (PE-102-1001) were used instead of those provided in the kit. For multiplexed samples, Illumina Multiplexing adapters and primers (PE-400-1001) were used. RNA-seq Iibraries were constructed with the standard procedure included with the Illumina TruSeq RNA Sample Preparation kit. ChIA-PET libraries were constructed with Illumina paired-end adaptors (PE-102-1001) and the protocol outlined in Fullwood, et al. 2010 (PMID:20069536)

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

ChIP-seq reads (E10.5 limb CTCF) were aligned to mm9 genome assembly using Bowtie (0.12.3) and only uniquely aligning reads were retained (bowtie -B 1 -p 8 --solexa1.3-quals). To call peaks, MACS (1.3.7) was supplied with both ChIP and input samples (input from GSE 30641) and run with the following parameters: --format=BOWTIE --tsize=74 --gsize=1860000000 --bw=150 --pvalue=0.00001 --mfold=10.
ChIP-seq reads (E11.5 limb CTCF, Smc1a, H3K27ac, H3K4m2, H327me3) were aligned to mm9 genome assembly using Bowtie (0.12.7) and only uniquely aligning reads were retained. For single-end sequencing data, the following parameters were used: bowtie -B 1 -p 8 -m 1. For paired-end sequencing reads, an additional parameter was used (-X 1000). To call peaks for Smc1a and CTCF, MACS (1.4.1) was supplied with both ChIP and input samples and run with the following parameters: macs14 --pvalue=0.00001 --bw=300 -f BOWTIE -g mm. For CTCF, an additional parameter (mfold=5,30) was used. For calling peaks with paired-end aligned reads, only the first mate pair was used for both the ChIP and input samples. To call peaks for H3K27ac, H3K4me2, and H3K27me3, a custom perl script was implemented based on methodology from Mikkelsen et al. 2010 (PMID 20887899).
RNA-seq reads were aligned to the mm9 genome assembly using TopHat (v1.4.1) and supplying a known transcriptome index from UCSC (tophat -p 8 -g 1 --transcriptome-index=/home/GENOMES/transcriptome/mm9_known).
ChIA-PET reads were trimmed of ChIA-PET ligation adaptors using Cuadapt (0.9.4) and the following parameters: cutadapt -aAGTTGGATACCTGCAGTACTAGTCAGTGGGCCC -m16 -M16 -O33. Trimmed mate-pairs were then aligned to the mm9 genome assembly using ELAND (CASAVA v1.8.1) with the following parameters: -ub Y\* --bam. SAMtools was used to filter only reads with MAPQ ≥ 1 for downstream analysis. Aligned reads were paired with mates and were filtered for PCR duplicates (>1 paired-end read with same start positions of both mates). Paired-end reads were categorized into interchromosomal, intrachromosomal (distance between mates > 5kb), and self (distance between mates < 5kb). Only intrachromosomal reads with mates less than 1Mb apart and overlapping at least 1 Smc1a ChIP-seq peak were considered.
Genome_build: mm9
Supplementary_files_format_and_content: ChIP-seq and ChIA-PET samples are supplied with BED files containing coordinates of either ChIP-seq peaks or ChIA-PET interaction intervals. Wig files were generated for RNA-seq and ChIP-seq samples, normalized by sequencing depth, and converted to bigWig with the wigToBigWig utility.

Submission date

Nov 13, 2012

Last update date

May 15, 2019

Contact name

Laura DeMare

E-mail(s)

laura.demare@yale.edu

Organization name

Yale University School of Medicine

Department

Genetics