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| Status |
Public on Apr 09, 2013 |
| Title |
Histone mark #1-4: input (Nkx2-1-pos. tumor), rep. #2 |
| Sample type |
SRA |
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| Source name |
lung adenocarcinoma
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| Organism |
Mus musculus |
| Characteristics |
tester-control pair: H
strain: mixed 129/B6
tissue: lung
tumor type: adenocarcinoma
nkx2-1 status: positive
gender: female
age: 4 to 7 months
chip antibody: n/a
chip antibody catalog number: n/a
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| Treatment protocol |
Mice were infected intratracheally with adenovirus (University of Iowa, Gene Transfer Vector Core) or lentivirus as described (DuPage et al., 2009). Tamoxifen (Sigma) was dissolved in corn oil (Sigma) and administered intraperitoneally at 56 micrograms (low dose) or 112 micrograms (high dose) per gram total body weight.
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| Growth protocol |
n/a
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lung tumors were pulverized, cross-linked with 1% formaldehyde in PBS for 10 min at RT, washed in 5mg/ml BSA in PBS and then in just cold PBS, re-suspended in lysis buffer [50mM Tris-HCl, pH 8.1, 10mM EDTA, 1% SDS, 1X complete protease inhibitors (Roche)], and sonicated with the Covaris E210 sonicator to obtain chromatin fragment lengths of 200-to-1500bp judged by Bioanalyzer DNA High sensitivity kit (Agilent). Fragmented chromatin was diluted in IP buffer [20mM Tris-HCl pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100] and incubated overnight at 4C with Protein G magnetic beads (Dynabeads: Invitrogen) that had been pre-incubated with anti-NKX2-1 (Bethyl BL4000), anti-FOXA1/2 (Santa Cruz sc-6553), H3K4me1 (Abcam ab8895), H3K4me3 (Millipore 07-473), H3K27Ac (Abcam ab4729) or H3K27me3 (Millipore 07-449) antibodies. Immunoprecipitates were washed six times with wash buffer [50mM HEPES pH 7.6, 0.5M LiCl, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40] and twice with TE buffer. Immunoprecipitated (or no IP input) DNA was treated with RNase A and Proteinase K on the beads, recovered in 1% SDS plus 0.1M NaHCO3 over a period of 5 hours at 65C, column purified with QiaQuick columns (Qiagen) and quantified with use of a PicoGreen assay (Invitrogen). After a sonication to enrich DNA fragment lengths between 100-300bp, 5 to 50ng of DNA were used for the library construction.
After a sonication to enrich DNA fragment lengths between 100-300bp, 5 to 50ng of DNA were used for the library construction. DNA sequencing for Illumina cluster generation were prepared using the SPRI-works system (Beckman Coulter) with 200-400 bp size selection followed by enrichment with barcoded PCR primers for multiplexing at the MIT BioMicro Center. Sequencing was performed on a HiSeq2000 (HiSeq Flow Cell v3, TruSeq SBS Kit v3, RTAVersion 1.13.48) running HiSeq Control Software 1.5.15.1 for 40 to 50 nucleotides from a single end. Barcode-separated FASTQ files were generated from QSEQ files using a custom parsing script.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Forty nucleotide of sequenced reads were mapped to reference mouse genome build 37 (mm9), using bowtie aligner. Aligned reads for two biological replicates for each IP and input.were merged.
Sequencing reads from one Nkx2-1 IP were down-sampled to obtain equal contributions from each replicate.
Binding sites for Nkx2-1, and Foxa1/2 were identified using MACS (version 1.4.2) (Zhang et al. 2008 PMID:19505939), with a p-value cutoff of 10^-6 and with default values for other parameters.
H3K4me1, H3K4me3, H3K27Ac and H3K27me3 modified regions were detected using SICER (version 1.1) (Zang et al. 2009 PMID:19505939) with an effective genome fraction of 0.77, a FDR cutoff for calling peak regions (“islands”) of 0.01, and a window size of 200. Following authors’ recommendations, a gap size of 200 (600) was used for H3K27ac and H3K4me3 (H3K27me3 and H3K4me1).
Genome_build: mm9
Supplementary_files_format_and_content: .wig: alignment wiggle files of paired replicates, rescaled to a target of 40 Mio // .bed: called peaks, based on paired replicates
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| Submission date |
Jan 02, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Sebastian Hoersch |
| Organization name |
Massachusetts Institute of Technology
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| Department |
David H. Koch Institute for Integrative Cancer Research at MIT
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| Lab |
Bioinformatics and Computing Core
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| Street address |
77 Massachusetts Avenue (E18-366)
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02474 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE43252 |
Nkx2-1 Represses a Latent Gastric Differentiation Program in Lung Adenocarcinoma |
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| Relations |
| SRA |
SRX213855 |
| BioSample |
SAMN01881930 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1059374_tumPOS_input.mrgd.m142_treat_afterfiting_all.rscld40M_21.wig.gz |
378.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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