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| Status |
Public on Jan 31, 2016 |
| Title |
PC9_EZH2inh_H3K4me3 |
| Sample type |
SRA |
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| Source name |
NSCLC
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| Organism |
Homo sapiens |
| Characteristics |
cell line: PC9
chip antibody: H3K4me3 (CST 9751 Lot 7)
treatment: EZH2i
timepoint: 5days
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| Treatment protocol |
Cells were treated with 1 μM EZH2 inhibitor GSK126 in the same media with 5% FBS for 5 days
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| Growth protocol |
PC9 cells were cultured at 37 °C with 5% CO2 in RPMI-1640 (Invitrogen) supplemented with 10% FBS (HyClone) and antibiotics (Invitrogen). Drosophila Schneider (S2) cells were cultured at 28 °C without CO2 in Schneider’s medium (04-351Q, Lonza) containing 1% FBS. OSS Drosophila cells cultured as described (Niki et al. 2006)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. For each ChIP reaction, 30 ug of chromatin was precleared with protein A agarose beads (Invitrogen). ChIP reactions were set up using precleared chromatin and antibodies and incubated overnight at 4 C. Protein A agarose beads were added and incubation at 4 C was continued for another 3 hr. Immune complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on HiSeq 2500 (50 nt reads, single end).
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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 |
50-nucleotide sequence reads were aligned to the hg19 genome using the BWA algorithm with default settings. Only reads that passed Illumina’s purity filter, aligned with no more than 2 mismatches and mapped uniquely to the genome, were used in subsequent analyses.
The aligned read files were sorted and duplicates removed using sort and rmdup functions from samtools version 0.1.18 (Li et al., 2009).
WIG files were generated using IGVTools (version 2.2.2) count function with a window size of 25 nucleotides, an extension of 100 beyond the 50-nucleotide read length, and genome hg19 (Robinson et al., 2011; Thorvaldsdottir et al., 2013). The WIG files were scaled assuming an effective genome length of 2.79B base pairs, so that the mean signal would be 1.0.
Genome_build: hg19
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| Submission date |
Dec 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Suchit Jhunjhunwala |
| E-mail(s) |
suchitj@gene.com
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| Organization name |
Genentech
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| Street address |
1 DNA Way, MS-444A
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| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE64243 |
A ChIP-seq spike-in method enables detection of global histone modification changes across the genome |
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| Relations |
| BioSample |
SAMN03267952 |
| SRA |
SRX815739 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1566896_16_PC9_EZH2inh_H3K4me3_Dmspike_hg19_i89_dmnorm-W200-G200-E1.scoreisland.bed.gz |
292.0 Kb |
(ftp)(http) |
BED |
| GSM1566896_16_PC9_EZH2inh_H3K4me3_Dmspike_hg19_i89_stdnorm-W200-G200-E1.scoreisland.bed.gz |
315.6 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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