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| Status |
Public on Nov 01, 2015 |
| Title |
Lung_HZ54_H3K27me3 |
| Sample type |
SRA |
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| Source name |
Lung
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| Organism |
Mus musculus |
| Characteristics |
tissue: lung tumor
genotype: EZH2 overexpressing
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| Growth protocol |
Fresh murine lung tumor nodules were minced and cultured in 100-mm dishes with RPMI-1640/10% FBS/1% penicillin–streptomycin. H661 and H292 cells (obtained from American Type Culture Collection) were cultured in RPMI-1640/10% FBS/1% penicillin–streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mouse lung tissue was pulverized using Covaris Tissue Smasher model CP02 by following the CryoPrep Dry Pulverization Manuel. Lung tissue was smashed 1-2 times on setting 4 in the tissueTUBE (Covaris #520071). Approximately 50mg of pulverized lung tissue was cross-linked with prewarmed 1% formaldehyde (ThermoScientific #28906 diluted in PBS) for 20 minutes at 37°C. The tissue was spun down at 1,000rpm for 2 minutes and quenched with 0.125M glycine in PBS + 0.5% BSA for 20 minutes at room temperature, spun down at 1,000rpm for 2 minutes and washed with PBS + 2x Protease Inhibitor Cocktail (PIC) (Roche #11873580001) + 5mM Sodium Butyrate (Millipore #19-137) then spun down at 1,000rpm for 2 minutes. The cross-linked tissue was then lysed with 390uL ChIP Lysis Buffer (1% SDS, 10nM EDTA pH8.0, 50mM Tris-HCl pH 8.0, 2x PIC and 5mM Sodium Butyrate) on ice for one hour. The lysate was split into 3 microTUBEs (Covaris #520045) and sheared on the Covaris E210 Series with 5% Duty Cycle, 5 Intensity, 200 Cycles per Burst for a total of 27 minutes. The sheared chromatin was spun down at 14,000rpm for 15 minutes at 4°C. An aliquot of input was saved while the remaining chromatin was snap frozen and stored at -80°C. Input was brought up to 100ul with TE, 10ug of RNAseA (Roche) added and incubated for 30 minutes at 37°C followed by addition of 100ug of Proteinase K (Roche) and incubation at 65°C overnight. Input was purified with Qiagen PCR Purification Kit (#28104) and quantified
The prepared chromatin was thawed on ice while 10ug of antibodies against either H3K27ac (Abcam #Ab4729) or H3K27me3 (Cell Signaling #CS9733S) were conjugated to a mix of magnetic Protein A and Protein G coupled beads (Invitrogen #100.02D and #100.04D respectively) in the presence of 0.5% BSA in PBS with rotation at 4°C for 2 hours. Beads were washed 3 times with 0.5%BSA in PBS and either 5ug of chromatin was added to the H3K27ac ChIP or 10ug of chromatin was added to the H3K27me3 ChIP and rotated overnight at 4°C. The beads were washed 2 times with Tris based RIPA buffer (0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 7.4, 1mM EDTA pH 8.0, 0.1% Sodium Deoxycholate), 2 times with 0.3M NaCl RIPA (0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH 7.4, 1mM EDTA pH 8.0, 0.1% Sodium Deoxycholate, 0.3M NaCl), 2 times with LiCl Buffer (250mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate, 1mM EDTA pH 8.0, 10mM Tris-HCl pH 8.0) and 2 times with TE buffer pH 7.6 (Fisher Scientific cat. no. BP2474-1). The beads were resuspended in 100uL of TE and RNAseA and PK digested/reverse crosslinked and purified as described in the chromatin prep.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Chromatin IP against H3K27me3 in EZH2 overexpressing lung tumor
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| Data processing |
Images analysis and base calling was done using the solexa pipeline.
For all samples reads were aligned to their indicated build using bowtie with parameters -n 2 -e 70 -m 2 -k 2 --best -l 40. Seed length (-l) was set to read length for each dataset.
Genome_build: mm9
Supplementary_files_format_and_content: WIG files: For all samples aligned sequences processed through MACS 1.4.2 and fragment pileup was outputted in .wig format
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| Submission date |
Jun 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
James Bradner |
| E-mail(s) |
bradner_computation@dfci.harvard.edu
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| Organization name |
Dana-Farber Cancer Institute
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| Department |
Medical Oncology
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| Lab |
Bradner Lab
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| Street address |
450 Brookline
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE70045 |
Oncogenic deregulation of EZH2 as an opportunity for targeted therapy in lung cancer [ChIP-Seq] |
| GSE70047 |
Oncogenic deregulation of EZH2 as an opportunity for targeted therapy in lung cancer |
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| Relations |
| BioSample |
SAMN03783069 |
| SRA |
SRX1066703 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1716265_Lung_HZ54_H3K27me3.wig.gz |
75.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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