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| Status |
Public on Sep 18, 2012 |
| Title |
BrdU_RepliSeq S2 |
| Sample type |
SRA |
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| Source name |
cervical cancer cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa S3
chip antibody: BrdU
chip vendor: Becton Dickinson
chip catalog#: 347580
chip lot: 69138
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| Treatment protocol |
Formaldehyde was added to the culture medium to a final concentration of 1% (4 min at RT). Cross-linking was stopped by addition of glycine to a final concentration of 125 mM. Cells were washed twice with PBS and lysed in SDS buffer: 100 mM NaCl, 50 mM Tris HCl (pH 8.1), 5 mM EDTA (pH 8), 0.5% SDS and protease inhibitors. Chromatin lysate was then pelleted and washed once in IP buffer (100 mM NaCl, 100 mM Tris HCl (pH 8.1), 5 mM EDTA (pH 8), 0.3% SDS, 1.7% Triton X-100) and twice in sonication buffer (10 mM Hepes pH 7.6, 1 mM EDTA, 0.5 mM EGTA, protease inhibitors). The cells were resuspended in sonication buffer to a concentration of 3x107 cells/ml. Sonication, sample preparation for equilibrium density centrifugation, dialysis of collected fractions and analysis of DNA or protein content of each fraction were performed as previously described (Schwartz et al. 2005), with minor modifications. The sample volume was adjusted to 12 ml before centrifugation, transferred into a 14x89 mm Beckman Ultra-Clear centrifuge tubes and spun for 120 to 144 h at +20°C (34,000 rpm in a Sorvall S52-ST rotor). 750 μl fractions were collected with a peristaltic pump from the top of the tube.
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| Growth protocol |
HeLaS3 cells were grown by plating typically 300,000 cells/ml into a T75 flask in Dulbecco's Modified Eagle's Medium (Lonza) supplemented with South American Fetal Bovin Serum, Glutamine, penicillin and streptomicyn. For ChIP or IP assays cells were used at a concentration of 600,000-700,000 cells/ml
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP enriched or control DNA (1-10 ng) were blunt ended, 3'-dA overhangs were created by adding Klenow exo¯ with dATP and adapters were ligated. Unligated ADP was removed prior to PCR amplification (18 cycles)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Basecalls performed using CASAVA version 1.3
ChIP-seq reads were aligned to the hg18 genome assembly using bwa version 0.5.7 with the default parameters
H3K4me3 reads were trimmed to 36bps before alignment using fastx toolkit
Peaks were called using FindPeaks version 4.0.10 with the following setting: -dist type 1 (200), -qualityfilter (1), -duplicatefilter
To enhance signal-to-noise ratio from BrdU-positive regions (RepliSeq), data from all anti-BrdU IPs (S1-S6 windows and control) were processed with dspchip (v0.8.5, http://code.google.com/p/dspchip/). Control signal was subtracted from each S-phase window (S1-S6).
Genome_build: hg18
Supplementary_files_format_and_content: bedGraph files were generated using BEDtools (v 2.7.1)
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| Submission date |
Apr 25, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Lucilla Luzi |
| Fax |
+390294375991
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| Organization name |
IFOM FIRC Institute for Molecular Oncology Foundation
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| Street address |
Via Adamello 16
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| City |
Milano |
| ZIP/Postal code |
20139 |
| Country |
Italy |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE37583 |
Genome-wide mapping of human DNA-replication origins: levels of transcription at Orc1 sites regulate origin selection and replication timing |
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| Relations |
| SRA |
SRX144721 |
| BioSample |
SAMN00989389 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM922793_RepliSeq_S2_Hela_subtracted.bedGraph.gz |
112.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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