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| Status |
Public on Jan 21, 2020 |
| Title |
3T3_H3K27ac_pur input rep 2 |
| Sample type |
SRA |
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| Source name |
NIH3T3 cell culture
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| Organism |
Mus musculus |
| Characteristics |
strain background: NA
tissue/chip rxn: 1x10^6 cells/ChIP
age: NA
chip antibody: None
genotype or treatment: 48 hours LIF + 400 nm purmorphamine
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| Treatment protocol |
NIH3T3 cells were grown in 60 mm plates at 37°C and 5% CO2 in DMEM (ATCC 30-2002) supplemented with 10% Calf Serum (ATCC 30-2030), 1x L-Glutamine (Gibco 25030-08), 100 uM non-essential amino acids (NEAA from Gibco 11140-050), and 50 ug/mL Pen/Strep (Gibco 15140-122). For Hedgehog pathway stimulation, cells were switched to low serum media (0.5% calf serum) 24 hours prior to treatment with 400nM Purmorphamine (Stemgent cat # 04-0009). Cells were incubated in the presence of Purmorphamine for 48 hours then collected for ChIP.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed for 15 minutes at room temperature with 1% formaldehyde then lysed. After cell lysis, chromatin was sheared in shearing buffer containing 0.25% SDS using a Covaris S2 focused ultrasonicator. Sheared chromatin was incubated with antibody coated dynabead preparations (1ug antibody with 20ul beads) overnight. Beads were washed 5 times with RIPA buffer (1% NP40, 0.7% Sodium Deoxycholate, 1mM EDTA pH8, 50mM Hepes-KOH pH7.5, 2% w/v Lithium Chloride) and then eluted at 70°C for 15 minutes in elution buffer (50mM TRIS pH 8, 10mM EDTA pH 8, 1% SDS). Crosslinks were reversed overnight at 70°C and chromatin was purified and concentrated by phenol chloroform extraction and EtOH precipitation.
One third of each ChIP sample was used for library preparation. Library preparation was done according to the Broad Pond method, (PMCID: PMC3091298) using the NEBNext library preparation kit (NEB E6040L) using non-standard 15 cycles of PCR amplification.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Base calling was done using Illumina bcl2fasq version 1.8.4 or version 2.17.1
Raw sequencing reads are mapped to mouse mm10 genome using Bowtie2 (version 2.2.5).
Peak calling was done using Cisgenome with default parameters. When available, Input data was incorporated in the peak calling.
For differential H3K27ac and H3K4me2 peak analysis and peak classifications, Cisgenome was used to call peaks for Wild Type and Shh-/- (or control and Purmorphamine treated NIH3T3 cells) respectively. For the union of peaks from Wild Type and Shh-/-, we calculated the number of H3K27ac reads for WT H3K27ac, WT input, Shh-/- H3K27ac and Shh-/- input samples that overlap with each of the union peaks. The read counts are then divided by library size and log2 transformed after adding a pseudo count of 1 to generate the normalized read counts. We use Limma [2] to perform differential analysis in order to independently compare WT H3K27ac vs WT input, Shh-/- H3K27ac vs Shh-/- input, and WT H3K27ac vs Shh-/- H3K27ac to obtain adjusted p-values.
Genome_build: mm10
Supplementary_files_format_and_content: bedgraph files: aligned sequencing reads to mm10 genome using Bowtie2 and converted to bedgraph
Supplementary_files_format_and_content: txt files: peaks called by Cisgenome
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| Submission date |
Nov 15, 2017 |
| Last update date |
Jan 21, 2020 |
| Contact name |
Steven Vokes |
| E-mail(s) |
svokes@austin.utexas.edu
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| Organization name |
The University of Texas at Austin
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| Department |
Department of Molecular Biosciences, Institute for Cellular & Molecular Biology
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| Street address |
2500 Speedway, Stop A4800
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| City |
Austin |
| State/province |
Texas |
| ZIP/Postal code |
78712 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE106948 |
Genome wide maps of chromatin modification in response to Hh signaling in mouse embryonic forelimb buds |
| GSE108880 |
GLI transcriptional repression regulates enhancer activity and chromatin accessibility for Hedgehog target genes |
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| Relations |
| BioSample |
SAMN08028710 |
| SRA |
SRX3395810 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2858059_NIH3T3_H3K27ac_pur_input_rep2.bedgraph.gz |
312.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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