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| Status |
Public on Apr 01, 2017 |
| Title |
3565_culture_pol2 |
| Sample type |
SRA |
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| Source name |
Primary Patient Derived Tumor Model
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| Organism |
Homo sapiens |
| Characteristics |
cell type: 3565 cells
antibody: pol2
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| Treatment protocol |
cell isolation: Cells were collected
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| Growth protocol |
3565 cells from a primary patient derived GBM tumor model were injected intracranially into a immunocomprimised NSG mouse and allowed to grow into a tumor.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For histone modification and transcription factor ChIP-Seq, 2 million cells (H3K27Ac), 15-20 million cells (JMJD6), or 5 million cells (Pol2) were crosslinked in PBS + 1% fresh formaldehyde for 10 minutes at 37 C, quenched for 5 minutes with 125 mM glycine, washed twice in cold PBS with protease inhibitors (complete PI, Roche), and stored at -80 C. Pellets were thawed and lysed in cold cytoplasmic lysis buffer (20 mM Tris-HCl ph8.0, 85 mM KCl, 0.5% NP 40 + PI). Nuclei were pelleted at 3000g , resuspended in cold SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1 + PI) for 10 minutes, sonicated to an average fragment size of 200-400 bp on a Branson sonifier, and cleared of debris by centrifugation. Samples were diluted 1:10 in ChIP dilution buffer (0.01% SDS, 0.25% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl +PI), and rotated at 4 C overnight with 2-5 ug of antibody towards: H3K27ac (Active Motif, 39133), JMJD6 (Abcam, ab64575, lot GR54735-1), and total Pol II (Santa Cruz, sc-899-X, lot H0510). Antigen-antibody complexes were collected with protein G Dynabeads (Life technologies) for 4 hours at 4 C, and sequentially washed with RIPA buffer (0.1% Na deoxycholate, 0.1% SDS, 1% Triton x-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 140 Mm NaCl), RIPA/High salt (0.1% Na deoxycholate, 0.1% SDS, 1% Triton x-100, 10mM Tris-HCl pH 8.0, 1mM EDTA, 360 mM NaCl), LiCl Wash Buffer (250mM LiCl, 0.5% NP40, 0.5% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.0), and TE Buffer (10mM Tris-HCl pH 8.0, 1mM EDTA). Beads were resuspended in Low SDS ChIP elution buffer (10mM Tris-HCl pH 8.0, 0.5M EDTA, 300mM NaCl, 0.1% SDS, 5mM DTT) and incubated for 6 hours at 65 C to elute DNA and reverse crosslinking. Samples were treated with RNAase for 30 minutes and proteinase K for 2 hours at 37 C. ChIP DNA was then purified from supernatants with AMPure beads (Beckman-Coulter). Input DNA was prepared in parallel by adding unenriched, diluted chromatin directly into the elution / reverse crosslinking step.
ChIP DNA was end-repaired (End-It, Epicentre), A-tailed (Klenow fragment 3'-->5' exo-, New England Biolabs), and ligated to barcoded illumina adaptors (Quick T4 DNA ligase, NEB). Each reaction was followed by clean-up with AMPure beads (Beckman-Coulter). Ligation products were amplified by PCR for 14-18 cycles with illumina primers and PFU Ultra II HS PCR mix (Agilent). Library size selection to 300-600 bp was performed by two-step AMPure bead selection or gel purification (E-Gel SizeSelect 2%, Life technologies).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Sequencing of H3K27ac and JMJD6 ChIP and an unenriched chromatin control library (input) was performed on a NextSeq, with read lengths of 50bp
Reads were aligned to hg19 using BWA (Li, et. al., 2009)
Identical ChIP-seq sequence reads were collapsed to a single read to avoid PCR duplicates.
Peaks were called using HOMER v4.6 (Heinz, et. al., 2010) using matched inputs with the following parameters : H3K27ac, –histone –tagThreshold 50; JMJD6, –factor .
Genome_build: Hg19
Supplementary_files_format_and_content: Peak file (bed file) and bigwig file was generated for each sample by HOMER v4.6.
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| Submission date |
Oct 26, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Tyler E Miller |
| Organization name |
Cleveland Clinic Lerner Research Institute
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| Department |
Stem Cell Biology and Regenerative Medicine
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| Lab |
Jeremy Rich Lab
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| Street address |
2111 E 96th Street, NE3-256
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| City |
Cleveland |
| State/province |
Ohio |
| ZIP/Postal code |
44106 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE74520 |
Epigenetic Profile in Transcription elongation factors are in vivo-specific cancer dependencies in glioma |
| GSE74529 |
Transcription elongation factors are in vivo-specific cancer dependencies in glioma |
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| Relations |
| BioSample |
SAMN05944818 |
| SRA |
SRX2270414 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2360995_3565_culture_pol2.bigwig |
217.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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