NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1153766 Query DataSets for GSM1153766
Status Public on Sep 13, 2013
Title EBNA 3 ChIP-seq
Sample type SRA
 
Source name Mutu III Burkitt's Lymphoma cell-line
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
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.
Growth protocol Cells were cultured in RPMI 1640, 10% foetal bovine serum and antibiotics and passaged twice weekly.
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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer IIx
 
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)
 
Submission date Jun 04, 2013
Last update date Sep 13, 2013
Contact name Aditi Kanhere
E-mail a.kanhere@bham.ac.uk
Phone 0044-121-4145896
Organization name University of Birmingham
Street address School of Biosciences
City Birminghma
ZIP/Postal code B15 2TT
Country United Kingdom
 
Platform ID GPL10999
Series (1)
GSE47629 Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs epigenetic reprogramming
Relations
BioSample SAMN02190060
SRA SRX290878

Supplementary file Size Download File type/resource
GSM1153766_EBNA3C_peaks.txt.gz 100.7 Kb (ftp)(http) TXT
Raw data provided as supplementary file
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap