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Sample GSM1045814

Query DataSets for GSM1045814

Status

Public on Jan 18, 2013

Title

Prdm14KO_H3K27me3

Sample type

SRA

Source name

ES cells

Organism

Mus musculus

Characteristics

strain: C57BL/6
genotype/variation: Prdm14-/-
cell type: Mouse embryonic stem cells (mESCs)
chip antibody: H3K27me3 (Upstate/Millipore, 07-4492)

Growth protocol

ES cells were passaged in ESC maintenance medium [GMEM containing 2 mM L-glutamine (cat#11710-035; Invitrogen) with 1 × MEM non-essential amino acids (cat#11140-050; Invitrogen), 1 mM sodium pyruvate (cat#11360-070; Invitrogen), 100 M -mercaptoethanol (cat#21985-023; Invitrogen), 1 × 103 units/ml LIF (cat#ESG1107; Chemicon), and 20% FBS (Stem Cell Science)].

Extracted molecule

genomic DNA

Extraction protocol

ESCs at 70–80% confluency (approximately 2×107 cells on 15 cm dish) were crosslinked with 1% formaldehyde for 5 min at RT and rinsed twice with 10 ml of cold PBS after quenching with 125mM glycine. The fixed cells were harvested by using a silicon scraper, collected by centrifugation at 700 g for 5 min at 4 ○C, resuspended in 5 ml of cold LB1 (20 mM Tris, 10 mM NaCl, 2.5 mM MgCl2, 0.2% NP-40, 1 mM PMSF, pH7.5) and pelleted by centrifugation at 1350 g for 5 min after the rotation for 10 min at 4 ○C. The pellet was resuspended in 5 ml of LB2 (20 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, pH8.0) and rotated for 10 min at 4 ○C. The nuclei were pelleted by centrifugation at 1350 g for 5 min at 4 ○C and lysed in 2 ml of LB3 (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% TritoneX-100, 0.1% sodium deoxycholate, 0.1% SDS, CompleteTM EDTA-free). The lysed nuclei were sonicated using the following conditions: Branson Sonifier 250D with standard tip; 15% output; 10 cycles (12sec ON, centrifugation at 13,200 g for 2 min at 4 ○C).
Protein-DNA complexes were immunoprecipitated for overnight at 4 ○C using 10 μg of antibodies bound to 250 μl of Dynabeads Protein G (Invitrogen). Immunoprecipitates were washed five times with 1 ml of RIRA buffer (50 mM HEPES-KOH, 0.5 M LiCl, 1 mM EDTA, 0.5% Na-Deoxycholate, 1% NP-40, pH 7.4), and once with 250 μl of TE50 (50 mM Tris, 10 mM EDTA, pH8.0). Beads were resuspended with 200 μl of EB (1% SDS, TE50). The complexes were eluted at 65 ○C for 20 min. The crosslinks for both the immunoprecipitated and input DNA fragments were reversed by incubating at 65 ○C for at least 6 hrs. After RNase A and Proteinase K digestion, the DNA is purified by phenol/chloroform extraction and EtOH precipitation with 30 μg glycogen and dissolved with TE.
DNA from whole cell extracts (WCE) and ChIP fractions was further sheared to an average size of approximately 150 bp by ultrasonication (Covaris, Woburn, MA), end-repaired, ligated to sequencing adapters and amplified according to the manufacturer’s instructions (Applied Biosystems SOLiD Library Preparation Protocol).
For the ChIP of PRDM14 using AGP14 ESC-like cells, gel-purified amplified DNA of between 100 and 150 bp was sequenced on Life Technologies SOLiD 4 platforms to generate single-end 50-bp reads. For SUZ12 and histone H3K27me3, amplified DNA of between 200 and 350 bp, purified using an Agencourt AMPureXP Kit, was sequenced on a Life Technologies SOLiD 5500xl platform to generate single-end 50 bp reads.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

AB 5500xl Genetic Analyzer

Data processing

For the ChIP-seq of PRDM14, Reads were summed in 10 bp bins along the chromosome for ChIP and WCE, respectively. The read number of each bin was normalized with the total number of aligned reads for each chromosome. Scanning the genome with a 300 bp (30 bin) sliding window, a one-sided Wilcoxon rank-sum test was performed to estimate the enrichment p-value for each window. The fold enrichment (ChIP/WCE) for each window was also calculated. To call peaks, we picked up the windows which satisfied the criteria of a fold enrichment > 3.0 and p-value < 1e-4 as candidates for binding sites (Langmead et al., 2009). To further eliminate uncertain sites, we removed the regions with low ChIP reads using either of two criteria: 1) The average number of ChIP reads per region/the average number of ChIP reads in the genome < 3.0; 2) The maximum read intensity in ChIP bins was less than 50. We derived the consensus sequence motif by using MEME (Hu et al., 2011).
Genome_build: mm9

Submission date

Nov 29, 2012

Last update date

May 15, 2019

Contact name

Yukihiro Yabuta

E-mail(s)

yabyab@anat2.med.kyoto-u.ac.jp

Organization name

Kyoto University, Graduate school of medicine

Department

Anatomy and Cell Biology