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| Status |
Public on Apr 04, 2013 |
| Title |
S4 stage, repeat 2 |
| Sample type |
SRA |
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| Source name |
HeLa cell
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa cell
cell stage: S4 stage
molecule type: genomic DNA after ChAP pull-down
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| Treatment protocol |
Cells were either arrested by a thymidine-nocodazole block and released for 0 (mitosis), 4 or 7 (G1) hours or with a double thymidine block and released for 0, 2, 4, 6, 8, 10 or 12h. For transcription inhibition, HeLa-Ub cells were treated with flavopiridol (1µM) or α-amanitin (50µg/ml) for 3 and 5 hours respectively before crosslinking for ChAP.
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| Growth protocol |
HeLa cells were grown in DMEM supplemented with 10% BS, glutamax, penicillin/streptomycin and sodium pyruvate (Invitrogen). HeLa cells expressing the HBT tagged Ubiquitin (HeLa-Ub) (23)were grown in DMEM containing biotin (0.5 µM, Sigma Aldrich) and puromycin (1.5 ug/ml, Invitrogen).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin affinity purification (ChAP) samples for sequencing by Illumina GA II were prepared as follows. Chromatin affinity purification was based on a standard ChIP method (27) with modification of a two-step affinity purification. HeLa-Ub cells were cross-linked with 1% formaldehyde (Sigma) and the reaction stopped by adding 1/20 volume of 2.5 M glycine. The cross-linked material was then washed with PBS, lysed as for the ChIP protocol and sonicated to an average DNA fragment size of 200 bp. All buffers were freshly supplemented with 10 mM N-Ethylmaleimide (Sigma), 1 mM PMSF (Sigma), 1X Protease inhibitor cocktail (Sigma). The sheared chromatin was incubated with 375 µl Ni-NTA beads (Qiagen) for 16h at 4°C.An aliquot of the input DNA was saved prior to immunoprecipitation as reference sample. After washing in 6 ml of wash buffer I (50 mM Tris pH 8; 0.01% SDS; 1.1% Triton X-100; 150 mM NaCl), chromatin fragments were eluted in 3 cycles of 2 ml elution buffer I (50 mM Tris pH 8; 0.01% SDS; 1.1% Triton X-100; 150 mM NaCl; 300 mM Imidazole). The nickel eluate was incubated with 375 µl of avidin beads (Thermo Scientific) for 6h at 4°C. After washing in 1 ml of wash buffer II (50 mM Tris pH 8; 1% SDS; 1.1% Triton X-100; 1M NaCl) followed by two washes in low salt buffer (50 mM Tris pH 8; 1% SDS; 1.1% Triton X-100; 0.5 M NaCl), then two washes with 1 ml TE buffer (100 mM Tris pH 8; 10 mM EDTA; 50 mM NaCl). Crosslinks were reversed by adding 2 ml of elution buffer (50 mM Tris pH 8; 10 mM EDTA; 1% SDS; 200 mM NaCl) to the beads and incubating at 65°C for 15h. The supernatant was collected and diluted 1:2 with TE buffer. The eluate was treated with RNase (0.2 mg/ml final concentration; Sigma) for 2h at 37°C, followed by adding Proteinase K (0.2 µg/ml final concentration; Sigma) for 2h at 55°C, and DNA was extracted in phenol/chloroform/isomyl alcohol, and the DNA was precipitated in 200 mM NaCl (final concentration), 30 µg of glycogen (Ambion), 2x of the volume of ice cold 100% ethanol and incubation at -20°C for 1h followed by centrifugation. The pellet was washed in 500 µl of 70% ethanol, then the DNA was finally recovered, and its concentration was quantified by Picogreen assay (Invitrogen).
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303).
modified version of ChIP-seq using affinity tag (called ChAP-seq), so there is no antibody involved, we used His6-Biotin labeled ubiquitin for all ChAP-seq samples
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Cells were arrested by double thymidine block and released for 4 hours.
ChAP-Seq
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| Data processing |
Basecalls performed using CASAVA version 1.4
ChIP-seq reads were aligned to the hg18 genome assembly using ELAND
peaks were called using FindPeaks version 4.0.10 with the following setting: subpeaks 0.5, trim 0.2, dist_type 1 200, minimum 5-12, aligner bed, min_ht_process 5-12
Genome_build: hg18
Supplementary_files_format_and_content: txt file, contain peakcalling results generated by FindPeaks4.0.10
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| Submission date |
Apr 04, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jeffrey Parvin |
| URL |
http://parvinlab.bmi.ohio-state.edu/index.php?title=Main_Page
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| Organization name |
the Ohio State University
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| Department |
Biomedical Informatics
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| Street address |
904 Biomedical Research Tower
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| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43202 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE45771 |
Promoters Active in Interphase are Bookmarked during Mitosis by Ubiquitination |
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| Relations |
| SRA |
SRX259811 |
| BioSample |
SAMN01999040 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1114926_S4_C_min8_triangle_subpeaks.peaks.txt.gz |
2.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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