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| Status |
Public on Sep 13, 2013 |
| Title |
EBNA 3 ChIP-seq |
| Sample type |
SRA |
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| Source name |
Mutu III Burkitt's Lymphoma cell-line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: Mutu III
cell line origin: B-cell line derived from a tumour biopsy
clone number: clone 48
chip antibody: EBNA 3
chip antibody manufacturer: Abcam
chip antibody catalog #: ab16128
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| Treatment protocol |
Mutu III cells were diluted to 5x10 5 cells/ml 24 hrs prior to chromatin preparation. Cells were then resuspended at 1x10 7 cells/ml and formaldehyde was added to a final concentration of 1% (v/v) for 15 minutes at room temperature with rocking to cross-link proteins and DNA.
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| Growth protocol |
Cells were cultured in RPMI 1640, 10% foetal bovine serum and antibiotics and passaged twice weekly.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Following cross-linking, glycine was added to a final concentration of 1.25 M and cells were washed in PBS and lysed in 300 ul of cell lysis buffer (85 mM KCl, 0.5% NP-40, 5 mM PIPES pH 8.0, 1 mM PMSF and EDTA-free protease inhibitor cocktail (Roche)) per 10 7 cells. Following a 10 minute incubation on ice, cell nuclei were pelleted by centrifugation at 8000 rpm for 5 minutes at 4 oC in a bench-top Microfuge. The nuclei were then resuspended in 200 µl of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.0 plus protease inhibitors) per 10 7 cells and sonicated to reduce DNA length to between 200 and 600 bp. ChIP was carried out using 600 µl of chromatin from 30 x 10 6 cross-linked cells diluted 10-fold in IP dilution buffer and pre-cleared with protein A sepharose beads. An input control sample was removed and EBNA 3 proteins precipitated overnight with rotation at 4ºC using 60 µl sheep polyclonal anti-EBNA 3 antibodies (Abcam, ab16128). EBNA 2 was precipitated using 48 µg EBNA 2-specific mouse monoclonal antibody (PE2) followed by an additional 2 hr incubation with 80 µg rabbit anti-mouse antibodies (Dako). BSA-coated protein A sepharose beads were then added and immune complexes collected by rotation at 4ºC for 3 hrs. Beads were washed for 10 minutes at 4 ºC with rotation using a series of different wash buffers. Complexes were washed once in low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl), once in high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 500 mM NaCl), once in LiCl wash buffer (250 mM LiCl, 1% NP40, 1% Na deoxycholate, 1 mM EDTA, 10 mM Tris pH 8.0) and twice in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). Immune complexes were eluted in TE elution buffer (10 mM Tris pH 8.0, 5 mM EDTA, 1% SDS) at 65 ºC for 15 minutes. TE elution buffer was added to the input control sample and all samples were then incubated at 65 ºC overnight to reverse the crosslinks and samples were treated with 0.2 µg/ml RNAse A for 1 hr at 37 ºC to remove RNA. DNA was purified using QIAquick gel extraction (Qiagen).
ChIP and input DNA (10 ng) was used to generate sequencing libraries with a ChIP-seq sample prep kit (Illumina). DNA fragments were end repaired, phosphorylated, 3’-dA overhangs added and adapters ligated according to the manufacturer’s instructions. PCR-amplified samples (16-cycles) were separated on a 2% agarose gel in TAE buffer, visualized using SYBR-safe stain and a Dark Reader transilluminator (Clare Chemical Research). The region of the gel containing 150-350 bp DNA fragments was excised and DNA purified using a gel extraction kit (Qiagen). The library was quantified using an Agilent bioanalzer and subjected to 35bp single-end read sequencing with an Illumina Genome Analyzer IIx.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Image analysis and base calling were performed using the solexa pipeline. Reads were aligned to the human genome build hg19 using Eland, background corrected using whole cell extract data, and converted to tags per million total reads. Significant peaks were identified with MACS.
Genome_build: hg19
Supplementary_files_format_and_content: EBNA2_peaks.txt: peak file, MACS output (pval = 10-7)
Supplementary_files_format_and_content: EBNA3_peaks.txt: peak file, MACS output (pval = 10-7)
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| Submission date |
Jun 04, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Aditi Kanhere |
| E-mail(s) |
a.kanhere@liverpool.ac.uk
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| Organization name |
University of Liverpool
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| Street address |
Institute of Systems, Molecular and Integrative Biology
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| City |
Liverpool |
| ZIP/Postal code |
L69 3GE |
| Country |
United Kingdom |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE47629 |
Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs epigenetic reprogramming |
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| Relations |
| BioSample |
SAMN02190060 |
| SRA |
SRX290878 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1153766_EBNA3C_peaks.txt.gz |
100.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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