Splicing Factor ChIP and ChRIP: Detection of Splicing and Splicing Factors at Genes by Chromatin Immunoprecipitation

Pre‐mRNA processing begins as soon as the nascent RNA emerges from RNA polymerase II, linking nascent RNA and the factors that bind nascent RNA to chromatin. To determine where along the active genes processing factors accumulate in vivo, a splicing factor ChIP (SF‐ChIP) has been developed. SF‐ChIP is a variation on chromatin immunoprecipitation (ChIP), in which protein and nucleic acid complexes are crosslinked with formaldehyde and small fragments of DNA are immunopurified from the complex mixture. SF‐ChIP determines the location of splicing factors anywhere on chromatin; the analysis can be focused on genes of interest, or ChIP templates can be used as starting material for ChIP‐on‐chip or sequencing. In this chapter, the detailed methods are provided for SF‐ChIP in Saccharomyces cerevisiae and mammalian tissue culture cells, using quantitative PCR as a detection method. In addition, details are provided of a second method – chromatin RNA immunoprecipitation (ChRIP) – which enables the detection of RNA processing intermediates through the immunopurification of nascent RNA associated with chromatin. In ChRIP, living cells are also crosslinked with formaldehyde, and the immunopurification of chromatin with antibodies specific for active histones is followed by the isolation of RNA and quantitative RT‐PCR. Both of these methods are used widely to determine which RNA processing events are cotranscriptional, and how recruitment of RNA processing factors to nascent RNA is achieved in the cell.