Simon Matthew D
Department of Molecular Biophysics and Biochemistry and Chemical Biology Institute, Yale University, West Haven, Connecticut, USA.
Curr Protoc Mol Biol. 2013;Chapter 21:Unit 21.25.. doi: 10.1002/0471142727.mb2125s101.
The genome is regulated by trans-acting factors that bind to specific loci in chromatin. In addition to protein factors, it has become clear that large non-coding RNAs can also act on chromatin at sites distant from where they are transcribed. This unit describes a means of identifying the genomic targets of those large non-coding RNAs. To accomplish this, the endogenous RNA of interest (here Drosophila roX2 is used as an example) is enriched from cross-linked chromatin extracts using short biotinylated complementary oligodeoxyribonucleotides. The targets of the RNA can be determined by examining the proteins and DNA that are enriched under these conditions. This analysis can be extended genome-wide by subjecting the enriched DNA to deep sequencing.
基因组由与染色质中特定位点结合的反式作用因子调控。除了蛋白质因子外，越来越清楚的是，大型非编码RNA也可以在远离其转录位点的区域作用于染色质。本单元描述了一种鉴定那些大型非编码RNA基因组靶点的方法。为实现这一点，使用短的生物素化互补寡脱氧核糖核苷酸从交联染色质提取物中富集感兴趣的内源性RNA（这里以果蝇roX2为例）。通过检查在这些条件下富集的蛋白质和DNA，可以确定RNA的靶点。通过对富集的DNA进行深度测序，这种分析可以扩展到全基因组范围。
