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| Status |
Public on Aug 19, 2015 |
| Title |
macroH2A1 OIS ChIP replicate 1 |
| Sample type |
SRA |
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| Source name |
IMR90 primary lung fibroblasts_macroH2A1_IP
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: macroH2A1 (Millipore, 07-219)
cell line: IMR90
cell type: primary fetal lung fibroblast
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| Treatment protocol |
Cells were infected with retrovirus expressing pBABE-RASV12 and selected with puromycin (1µg/ml) for 14 days
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| Growth protocol |
IMR90 (ATCC, CCL-186) primary human fetal lung fibroblast cells were cultured in MEM supplemented with 10% fetal bovine serum.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked with 1% formaldehyde in PBS at room temperature for 10 min, then quenched in 125 mM glycine in PBS at room temperature for 5 min. The cells were washed by cold PBS once and were collected by centrifugation and sonication in lysis buffer (50mM Tris pH 7.9, 10 mM EDTA, 1% SDS, 1x protease inhibitor cocktail and 1mM DTT) to generate chromatin fragments of ~500 bp in length. The material was clarified by centrifugation 14,000rpm for 10min at 4 °C, and 20 µl supernatant was used as input for quantitation, the remaining supernatant was diluted 10-fold in dilution buffer (20 mM Tris pH 7.9, 2 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 1X protease inhibitor cocktail and 1mM DTT), and pre-cleared by incubation with protein A-agarose beads at 4°C for 3hr. The pre-cleared, chromatin-containing supernatant was used in immunoprecipitation reactions at 4°C overnight with an antibody against macroH2A1, then incubated with protein A-agarose beads at 4°C for 2hr. No antibody negative controls were included for every ChIP experiment. The immuneprecipitated DNA was cleared of protein by digestion with 0.4mg/ml glycogen and proteinase K (2.5U/ml, Roche) in Txn stop buffer (20 mM EDTA, 0.2 M Nacl and 1% SDS) at 37°C for 1 hour. The DNA was then extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitated.
DNA fragments from ChIP reactions were repaired using T4 DNA Polymerase (NEB) and T4 DNA Polynucleotide kinase (NEB) in T4 DNA ligase buffer (NEB). Next, dA ends were generated by employing Klenow Fragment (3’->5’ exo-, NEB), in Buffer 2 (NEB). Adapter ligation reactions were then set up using indexed adapters and Quick Ligase (NEB). Ligation-mediated PCR was then performed using Phusion High-Fidelity DNA Polymerase (NEB). The libraries were visualized on 2% E-Gel® General Purpose Agarose Gels (Invitrogen). Sequencing for the macroH2A1 ChIPs were performed on an Illumina HiSeq 2500 at the Einstein Epigenomics Core Facility. Sequences reads were mapped to the human genome version hg19 using ELAND version 1.7.0 (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Basecalls performed using CASAVA version 1.7
ChIP-seq reads were aligned to the hg19 genome assembly using ELAND version 1.7.0
significant genomic regions were identified by comparing matched ChIP and input samples with ISOR version 0.4 with the following settings: segmentation_pval(0.01), gap_length(1000)
Genome_build: hg19
Supplementary_files_format_and_content: gappedPeak files are as described (http://genome.ucsc.edu/FAQ/FAQformat.html#format13) the signalValues represent the log2 odds ratio of a segment and the pValue (-log10) represents the significance of the enrichment or depletion. The gaps are regions of at least 1kb with no mapped reads from either the input or ChIP samples; bedGraph files for segments where gaps have been subtracted. The scores represent the log2 odds ratio of each segment.
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| Submission date |
Jan 02, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew Jon Gamble |
| E-mail(s) |
matthew.gamble@einstein.yu.edu
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| Organization name |
Albert Einstein College of Medicine
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| Department |
Molecular Pharmacology
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| Lab |
Gamble Lab
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| Street address |
1300 Morris Park Ave / Golding 203
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| City |
Bronx |
| State/province |
NY |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE64601 |
MacroH2A1 ChIP-seq from H-RasV12 expressing senescent IMR90 human primary lung fibroblasts |
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| Relations |
| BioSample |
SAMN03274461 |
| SRA |
SRX826360 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1575698_m1.In.110121.P0.01.G.1000.gappedPeak.gz |
1.1 Mb |
(ftp)(http) |
GAPPEDPEAK |
| GSM1575698_m1.In.110121.P0.01.G.1000.gappedSegments.bedGraph.gz |
644.7 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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