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Curr Protoc Microbiol. 2016 May 6;41:1E.11.1-1E.11.18. doi: 10.1002/cpmc.3.

CLIP-seq to Identify KSHV ORF57-Binding RNA in Host B Cells.

Author information

1
Tumor Virus RNA Biology Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland.
2
Virus laboratory, Affiliated Shengjing Hospital of China Medical University, Shenyang, China.
3
Systems Biology Center, Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
4
Corresponding author.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is etiologically linked to the development of several malignancies, mainly Kaposi's sarcoma. Expressed as an early viral protein, KSHV ORF57 is essential for lytic replication and virion production. ORF57 selectively binds to a subset of viral RNA and affects nearly all aspects of viral RNA processing. To globally identify all viral and host RNA associated with KSHV ORF57 in the infected cells, we have utilized UV cross-linking and immunoprecipitation (CLIP) of KSHV ORF57 combined with high-throughput RNA sequencing (CLIP-seq) to identify ORF57-binding RNA in BCBL-1 cells at genome-wide level. This unit provides step-by-step details on this new method that is applicable for any pathogen or host RNA-binding proteins by slight modification.

KEYWORDS:

KSHV; ORF57; RNA; UV cross-linking; high throughput sequencing

PMID:
27153386
PMCID:
PMC6650155
DOI:
10.1002/cpmc.3
[Indexed for MEDLINE]
Free PMC Article

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