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Sample GSM1155709

Query DataSets for GSM1155709

Status

Public on May 14, 2014

Title

RBPJ in mitosis rep2

Sample type

SRA

Source name

F9_RBPJ_ChIP-seq

Organism

Mus musculus

Characteristics

cell line: F9
cell type: murine embryonic carcinoma cells; testicular teratoma
cell cycle stage: mitotic cells
antibody: anti-RBPJ antibody (custom-made)

Treatment protocol

Murine embryonic carcinoma F9 cells were harvested and fixed in 1% formaldehye, 10 min. Glycine was added to final 125mM 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 40% power amplitude, 30sec, 12 times total. 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, 70 μg of DNA was used to incubate with anti-RBPJ antibody (raised in our laboratory) or with same amount of 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 PCR purification kit.
ChIP protocol: 70 μg soluble chromatin was incubated with anti-RBPJ antibody (rasied in our laboratory) or with same amount of IgG as the negative control. BSA coated protein A agarous beads were used to obtain the ChIP product. Chromatin was reverse crosslinked at 65℃ 16hours, and DNA was purified by PCR purification kit.
For generating anti-mRBPJ antibodies, GST-mRBPJ was expressed in SF9 cells and purified using glutathione sepharose chromatography. The rabbit anti-RBPJ polyclonal antibody was generated by Cocalico Biologicals, Inc.

Growth protocol

DMEM, 10% FBS, 5% CO2

Extracted molecule

genomic DNA

Extraction protocol

After wash steps, cells were resuspended in lysis buffer and proceeded to the sonication with 40% power amplitude, 30sec, 12 times total. Cell debris was removed by 10 min centrifuge, 15,000 rpm at 4℃. Extracted chromatin supernatant was used for the following chromatin immunoprecipitation or frozen in -80℃ for storage.
To generate the library from ChIP experiemtn, 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/. According to the protocol, 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 defalt option
Genome_build: mm9
Supplementary_files_format_and_content: bed files were generated after peak calling

Submission date

Jun 07, 2013

Last update date

May 15, 2019

Contact name

Kyoung Jae Won

E-mail(s)

wonk@mail.med.upenn.edu

Organization name

University of Pennsylvania

Department

Genetics