|Public on Mar 18, 2015
|input plus acyclovir
|Akata BL cell line_with_acyclovir
|pathogen: Epstein-Barr virus
cell line: Akata
cell type: B lymphocytes
acyclovir dose: with 100uM acyclovir
antibody: none (input)
|0.125% rabbit anti-human IgG anti-IgG was added for 48 hours
|RPMI medium supplemented with 10% (v/v) fetal bovine serum, 100units/ml penicillin, 100μg/ml streptomycin and 2mM L-glutamine
|Chromatin was fixed by application of 1% (v/v) formaldehyde for 15 min then sonnicated on ice (10 x 10s-pulses; 30% amplitude output on a Branson model 250 Microtip at setting 5 (Sonics Vibacell). The chromatin was pre-cleared with protein A/G-sepharose bead slurry (Sigma) that had been pre-blocked in 0.5% (w/v) fraction V BSA (Sigma) in DPBS. 2% (v/v) of the pre-cleared extract was retained as the input control sample while the remainder was incubated with 10μg of Zta-specific goat antibody (Santa Cruz # sc-17503) or control goat IgG for 1 h at 4oC]. The input control sample and the precipitated DNA were sequentially treated with 0.2μg/ml RNAse A and 0.2μg/ml Proteinase K and the DNA purified.
Sequencing libraries were prepared using 10ng of the input and ChIP DNA using a ChIP-Seq sample preparation kit (Illumina) following the manufacturer’s protocol, except that the library (150-350bp fragments) was purified from the gel after PCR-amplification. The DNA fragments were separated on a 2% agarose gel in TAE buffer, visualized using SYBR-safe stain and 150-300bp fragments were excised and purified using a gel extraction kit (Qiagen).
|Illumina Genome Analyzer IIx
with 0.125% rabbit anti-human IgG anti-IgG added 48 hours
|Initial processing of sequencing images was carried out using the CASAVA pipeline. Base calling, and quality control statistics were performed using GOAT and Bustard modules.
Sequence reads were aligned to the hg19 release of the human genome with ELAND.
Bigwig files were generated by calculating tag density in 10-bp windows, normalizing per million total reads and subtracting the input signals using in-house R scripts.
Peak calling (p<10−7) was performed from a merged Bed file generated from the two ChIP datasets using MACS, with a merged input Bed file acting as background.
Supplementary_files_format_and_content: MACS output
|Jun 05, 2014
|Last update date
|May 15, 2019
|Alison Jane Sinclair
|University of Sussex
|School of Life Sciences
|Biochemistry and Bioscience
|University of Sussex
|Genomic-scale identification of host genes regulated by EBV during lytic cycle [ChIP-Seq]
|Genomic-scale identification of host genes regulated by EBV during the lytic cycle