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Status |
Public on Feb 02, 2017 |
Title |
ADC_H3K4me3_ChIPSeq |
Sample type |
SRA |
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Source name |
Lung tumors
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Organism |
Mus musculus |
Characteristics |
tissue: Lung tumors tumor subtype: Adenocarcinoma background: FSF:KrasG12D/+; FSF:R26:CreERT2; Lkb1flox/flox antibody: H3K4me3 (Millipore 07-473)
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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 or S2 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 4ºC with Protein G magnetic beads (Dynabeads: Life Technologies) that had been pre-incubated with 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 and 0.1M NaHCO3 over a period of 5 hours at 65ºC, column purified with DNA clean and concentrator -25 (Zymo Research). After a sonication to enrich DNA fragment lengths between 100-300bp, 5 to 10ng of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370). Sequencing was performed on a NextSeq (Illumina) for 38 nucleotides from paired ends according to manufacturer’s instructions.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NextSeq 500 |
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Description |
st_hw_Kras-Lkb1-Ad_1_K4me3
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Data processing |
ChIP-seq paired reads were aligned to the mm10 genome assembly using Bowtie2 with -k 1 option H3K4me3 and H3K27Ac peaks were called using MACS (v1.4.2) at p-value threshould of 1E-05 with no-ChIP input sample as control. Wiggle files with a 10bp resolution for H3K27me3 were generated by MACS v1.4.2 with tag shift and then rescaled to a larger of total number of uniquely alignable sequences in two datasets. Genome_build: mm10
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Submission date |
Feb 01, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Hideo Watanabe |
E-mail(s) |
hideo.watanabe@mssm.edu
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Organization name |
Icahn School of Medicine at Mount Sinai
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Department |
Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine
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Lab |
Hideo Watanabe
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Street address |
One Gustave L. Levy Place
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City |
New York |
State/province |
NY |
ZIP/Postal code |
10029 |
Country |
USA |
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Platform ID |
GPL19057 |
Series (1) |
GSE94365 |
Lkb1 inactivation drives lung cancer lineage switching governed by Polycomb Repressive Complex 2 |
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Relations |
BioSample |
SAMN06287500 |
SRA |
SRX2531387 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2474137_st_hw_kraslkb1ad_1_k4me3-p5_peaks.bed.gz |
382.9 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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