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| Status |
Public on Aug 04, 2015 |
| Title |
JEKO-1 H3K27ac |
| Sample type |
SRA |
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| Source name |
JEKO-1 H3K27ac
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| Organism |
Homo sapiens |
| Characteristics |
material type: Cell line
transgene: NA
treatment: NA
chip antibody: H3K27ac (39133, Active Motif)
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| Treatment protocol |
See individual samples
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| Growth protocol |
Cell lines were grown in IMDM + 20% FCS (Oci-Ly-7 and Oci-Ly-1) or RPMI + 20% FCS (all others)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For histone modification and transcription factor ChIP-Seq in cell lines, 10-20 million cells were crosslinked in growth media + 1% 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 the appropriate antibody. 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. For primary lymphoma samples, tumor cellularity from the frozen block was confirmed by H&E frozen section by a board-certified hematopathologist (RR), and the block was trimmed as needed to remove low tumor cellularity regions. 25 micron sections were then cut to a total of ~50 mg of tissue for each ChIP-Seq chromatin prep. Frozen sections were resuspended and dissociated in PBS + PI + 10 mM Na butyrate, formaldehyde was added to 1%, and crosslinking and quenching were performed as above. Subsequent steps were identical to those performed in cell lines except that 1) the cytoplasmic lysis step was omitted, and 2) following lysis in SDS lysis buffer, the sample was diluted 1:2 to 1:5 with ChIP dilution buffer prior to sonication, and to a final dilution of 1:10 afterwards. Prior to immunoprecipitation, DNA was quantified from sonicated chromatin, and an equivalent of 2-5 million cells was used per ChIP.
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 |
Paired-end sequencing of H3K27ac ChIP and an unenriched chromatin control library was performed on a HiSeq 2500 or NextSeq for all primary lymphoma samples and for 8 cell lines, with read lengths of 36 bp + 25 bp or 42 bp + 42 bp. Single-end sequencing was used for other samples.
Reads were aligned to hg19 using BWA.
The resulting alignment files were then filtered to remove PCR duplicates and reads mapping to >2 sites genome-wide
ChIP-Seq density tracks were generated with ‘igvtools count’ and visualized with IGV.
Genome_build: hg19
Supplementary_files_format_and_content: TDF density files were generated by igvtools
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| Submission date |
Jun 04, 2015 |
| Last update date |
Nov 01, 2021 |
| Contact name |
Yotam Drier |
| E-mail(s) |
yotam.drier@mail.huji.ac.il
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| Organization name |
The Hebrew University of Jerusalem
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| Department |
Immunology and Cancer Research
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| Lab |
Drier
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| Street address |
Faculty of Medicine, The Hebrew University, Ein Kerem
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| City |
Jerusalem |
| State/province |
Israel |
| ZIP/Postal code |
9112102 |
| Country |
Israel |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE69558 |
Detection of Enhancer-Associated Rearrangements Reveals Mechanisms of Oncogene Dysregulation in B-cell Lymphoma [ChIP-Seq] |
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| Relations |
| Reanalyzed by |
GSE186941 |
| BioSample |
SAMN03761445 |
| SRA |
SRX1048464 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1703903_jeko-1_h3k27ac_7572_p.tdf |
275.9 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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