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Sample GSM1642997

Query DataSets for GSM1642997

Status

Public on Jun 01, 2015

Title

Hopx E9.5 Hearts -EGS, Input

Sample type

SRA

Source name

E9.5 hearts

Organism

Mus musculus

Characteristics

tissue: embryonic heart
antibody: Input

Treatment protocol

Hearts from Hopx3Xflag/+ mouse embryos (E9.5) were harvested, pooled and fixed in 1% formaldehye, 10 min. Glycine was added to final 140mM to quench the fixation. Cells were pelleted and washed with PBS and wash buffers. After wash steps, cells were resuspended in lysis buffer and proceeded to the sonication with Diagenode Bioruptor (High power, 30 second cycles, 7.5 min total length X 3). Cell debris was removed by 10 min centrifuge, 15,000 rpm at 4°C. Extracted chromatin supernatant was used for the following chromatin immunoprecipitation or frozen in -80°C for storage. A portion of the chromatin supernatant was reverse crosslinked and total DNA was purified by PCR purification kit. According to the measurement from this pilot DNA quantification, 10 μg of DNA was used to incubate with anti-Flag antibody (Sigma M2) or with same amount of mouse IgG as the negative control. BSA coated protein A agarous beads were used to obtain the ChIP product. Chromatin was reverse crosslinked at 65°C ~16hours, and DNA was purified by Qiagen PCR purification kit.

Growth protocol

E9.5 embryonic hearts were harvested

Extracted molecule

genomic DNA

Extraction protocol

10 μg of DNA was used to incubate with anti-Flag antibody (Sigma M2) or with same amount of mouse IgG as the negative control. BSA coated protein A agarous beads were used to obtain the ChIP product. Chromatin was reverse crosslinked at 65°C ~16hours, and DNA was purified by Qiagen PCR purification kit.
After wash steps, cells were resuspended in lysis buffer and proceeded to the sonication with Diagenode Bioruptor (High power, 30 second cycles, 7.5 min total length X 3). Cell debris was removed by 10 min centrifuge, 15,000 rpm at 4°C. Extracted chromatin supernatant was used for the following chromatin immunoprecipitation or frozen in -80°C for storage.
To generate the library from ChIP experiements, ChIP-Seq Library Protocol with multiplexing was used. This protocol was developed before Illumina released its multiplex truSeq ChIP-seq kit, and the details can be found on http://ngsc.med.upenn.edu/. 10 ng of ChIP DNA product was used to start the library construction.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

ChIP-seq reads were aligned to the mm9 genome assembly using bowtie 0.12.7 using option of --best -v 2 --strata -m 1
Peaks were called using Homer with a FDR cutoff=0.01 and p-value=0.001. The peaks from two samples were merged when they overlap within 200bps
Genome_build: mm9
Supplementary_files_format_and_content: a merged bed file was generated after peak calling

Submission date

Mar 25, 2015

Last update date

May 15, 2019

Contact name

Kyoung Jae Won

E-mail(s)

[email protected]

Organization name

University of Pennsylvania

Department

Genetics