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| Status |
Public on Apr 11, 2016 |
| Title |
hCEC-OVOL2 |
| Sample type |
SRA |
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| Source name |
primary corneal epithelium cell
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| Organism |
Homo sapiens |
| Characteristics |
cell type: corneal epithelium cell
chip antibody: OVOL2
vendor: SantaCruz Biotech
cat. #: sc-85804
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| Treatment protocol |
Approximately 10^7 cells were cross-linked in formaldehyde (1% final concentration, 10 min at room temperature), and then quenched with glycine (5 min at room temperature). Fixed cells were lysed in 50 mM HEPES KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 alternative, 0.25% Triton supplemented with protease inhibitor at 4 °C (Roche, 04693159001), centrifuged at 950g for 10 min and re-suspended in 0.2% SDS, 10 mM EDTA, 140 mM NaCl and 10 mM Tris-HCl. Cells were then fragmented with a Covaris Sonicater (model E210) at 8 °C to a size range between 200–500 bp, and precipitated by centrifugation. Ten micrograms of each antibody were pre-bound by incubating with Protein-G Dynabeads (Invitrogen, 100-03D) in IP dilution buffer (16.7 mM Tris-HCl, pH 8.0, 1.2 mM EDTA, 1.1% Trion X-100, 167 mM NaCl, and 0.01% SDS). Washed beads were added to the chromatin lysate, and then incubated overnight at 4 °C. Samples were washed 2 times with IP dilution buffer, twice with Low NaCl buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 1% Trion X-100, 150 mM NaCl, and 0.1% SDS), twice with High NaCl buffer (20 mM Tris-HCl, pH8.0, 2 mM EDTA, 1% Trion X-100, 500 mM NaCl, and 0.1% SDS) twice with LiCl buffer (10 mM TE, 250mM LiCl, 0.5% NP-40, 0.5% DOC), twice with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA), and then eluted in 0.5% SDS, 5 mM EDTA, 25 mM Tris-HCl pH 8.0 with Pronase (Roche, 165921) at 42 °C for 2 h and at 65 °C overnight. And then DNA was purified with the MinElute Reaction Cleanup kit (Qiagen, 28204). Human-OVOL2 antibodies (SantaCruz Biotechnology, sc-85804) was used for chromatin immunoprecipitation experiments.
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| Growth protocol |
Human CEC cell line (Araki-Sasaki et al., 1995) was cultured in Dulbecco’s modified Eagle’s medium and Ham’s F12 (1:1 mixture) and included 10% fetal bovine serum, 5μg/mL insulin, 0.1 nmol/L cholera toxin, 10 ng/mL EGF.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
10 ng of immunoprecipitated DNA was ligated with adaptors of Illumina GAIIx using illumina TruSeq ChIP Sample Prep Kit (illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Sequence reads were mapped to the human genome HG38 by BWA aligner.
Genome_build: HG38
Supplementary_files_format_and_content: bigwig files were generated using bam_to_wiggle.py script (http://github.com/chapmanb/bcbb/blob/master/nextgen/scripts/bam_to_wiggle.py).
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| Submission date |
Mar 11, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Koji Kitazawa |
| E-mail(s) |
kkitazaw@koto.kpu-m.ac.jp
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| Organization name |
Kyoto Prefectural University of Medicine
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| Department |
Ophthalmology
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| Street address |
465 Kajiicho Kamigyoku
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| City |
Kyoto |
| State/province |
Kyoto |
| ZIP/Postal code |
601-0841 |
| Country |
Japan |
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| Platform ID |
GPL15520 |
| Series (2) |
| GSE67823 |
Master transcription factors in corneal epithelial cells |
| GSE72641 |
Master transcription factors in corneal epithelial cells [ChIP-seq] |
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| Relations |
| BioSample |
SAMN04547005 |
| SRA |
SRX1629831 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2086970_hCEC-OVOL2_sorted.bigwig |
193.7 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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