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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 18, 2008 |
| Title |
Normal Mouse IgG ChIP |
| Sample type |
SRA |
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| Source name |
Normal Mouse IgG ChIP
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| Organism |
Homo sapiens |
| Characteristics |
HeLa S3 cells (ATCC/National Cell Culture Center)
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| Growth protocol |
HeLaS3 cells were grown in Minimum Essential Medium modified for Suspension (SMEM, Invitrogen), supplemented with glutamine, 10% FBS, and antibiotics (penicillin-streptomycin) at 37C in 5% CO2, to a density of 5 x10^5 cells/ml.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HeLa S3 cells were fixed with 1% formaldehyde at room temperature for 10 min and the fixation was terminated by the addition of glycine to a final concentration of 125 mM. The fixed cells were washed in cold 1x Dulbecco’s PBS (Invitrogen) and swelled on ice in 10 ml hypotonic lysis buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 1 mM EDTA, pH 8.0, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, and protease inhibitors). Cell lysates were homogenized with 30 strokes in a Dounce homogenizer. Nuclear pellets were collected and lysed in 10 ml of 1x RIPA buffer per 3 x108 cells (1x RIPA buffer: 10 mM Tris-Cl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid, 0.5 mM PMSF, 1 mM DTT, and protease inhibitors). Chromatin was sheared with a Branson 250 Sonifier (power setting 2, 100% duty cycle for 7x 30 s intervals) to an average size of less than 500 bp as verified on a 2% agarose gel. Lysates were then clarified by centrifugation for 15 min at 20,000 xg at 4°C. For normal mouse IgG ChIP samples, 12 mg of normal mouse IgG (Santa-Cruz sc-2025) were added to 1 x108 cells. Chromatin immunoprecipitations were conducted as previously described (Hartman et al. 2005; Euskirchen et al. 2007). The IgG ChIP DNA samples were run though Qiagen MinElute PCR columns, eluted with 15 μl of Qiagen buffer EB and size-selected (100-350 bp) on 2% agarose E-gels (Invitrogen). Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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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 |
size selected, normal mouse IgG ChIP DNA from HeLa S3 cells
size selected for 100-350 bp
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| Data processing |
Raw data from the Illumina Genome Analyzer and Illumina Genome Analyzer II were analyzed with Illumina’s Firecrest, Bustard and GERALD modules for image analysis, basecalling and run metrics respectively. Replicates were combined for each sample. All reads were aligned against human genome build hg18. The PolII, Sono-Seq (large fragment, small fragment, and interferon-gamma treated), and normal IgG were scored relative to naked DNA using PeakSeq (see "Peak-Seq - Systematic Scoring of ChIP-Seq Experiments Relative to Controls by Rozowsky et al. Nature Biotechnology. In press). Signal files were created using a 200 bp sliding window for all samples other than Sono-Seq/Input DNA-Large Fragment (575 bp). Two peak lists were created for each sample: an unfiltered list of all hits with p-value < 0.05 and a subset of this list that was filtered based upon sample tag count and enrichment versus naked DNA. Cutoff values were scaled for each sample relative to the number of reads. For PolII, normal mouse IgG, and Sono-Seq/Input DNA (small fragments), we required all peaks to have at least 20 sample tags and an enrichment of at least 5.0 versus the naked DNA reference. For Sono-Seq (interferon-gamma stimulated), cutoffs of 16 sample tags and 4.0x enrichment were used. For Sono-Seq (large fragments), cutoffs of 13 sample tags and 3.0x enrichment were used. MNase-digested DNA was not scored.
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| Submission date |
Dec 17, 2008 |
| Last update date |
May 15, 2019 |
| Contact name |
Raymond K Auerbach |
| Organization name |
Yale University
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| Street address |
266 Whitney Ave
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06511 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (1) |
| GSE14022 |
Mapping Novel Chromatin Regions Using Sono-Seq |
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| Relations |
| SRA |
SRX012413 |
| BioSample |
SAMN00004745 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM352186_IgG_mouse_from_HeLa_eland_results_rep1_laneA_FC309CE_20080818_s_3.txt.gz |
177.9 Mb |
(ftp)(http) |
TXT |
| GSM352186_IgG_mouse_from_HeLa_eland_results_rep1_laneB_FC309CE_20080818_s_4.txt.gz |
146.6 Mb |
(ftp)(http) |
TXT |
| GSM352186_IgG_mouse_from_HeLa_eland_results_rep1_laneC_FC309CE_20080818_s_5.txt.gz |
144.6 Mb |
(ftp)(http) |
TXT |
| GSM352186_IgG_mouse_from_HeLa_eland_results_rep2_laneA_FC30CNU_20080830_s_2.txt.gz |
140.4 Mb |
(ftp)(http) |
TXT |
| GSM352186_IgG_mouse_from_HeLa_eland_results_rep2_laneB_FC30CNU_20080830_s_3.txt.gz |
284.8 Mb |
(ftp)(http) |
TXT |
| GSM352186_IgG_mouse_from_HeLa_eland_results_rep2_laneC_FC30CNU_20080830_s_4.txt.gz |
110.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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