Analysis of long non-coding RNAs produced by a specialized RNA polymerase in Arabidopsis thaliana

Long non-coding RNAs (IncRNAs) play important roles in several processes including control of gene expression. In Arabidopsis thaliana, a class of IncRNAs is produced by a specialized RNA Polymerase V (Pol V), which is involved in controlling genome activity by transcriptional gene silencing. IncRNAs produced by Pol V have been proposed to serve as scaffolds for binding of several silencing factors which further mediate the establishment of repressive chromatin modifications. We present methods for discovery and characterization of IncRNAs produced by Pol V. Chromatin Immunoprecipitation coupled with deep sequencing (ChIP-seq) allows discovery of genomic regions bound by proteins in a manner dependent on either Pol V or transcripts produced by Pol V. RNA Immunoprecipitation (RIP) allows testing IncRNA-protein interactions at identified loci. Finally, real-time RT-PCR allows detection of low abundance Pol V transcripts from total RNA. These methods may be more broadly applied to discovery and characterization of RNAs produced by distinct RNA Polymerases. (C) 2013 Elsevier Inc. All rights reserved.