Molecular protocol for genome-wide and cell-type-selective profiling of in vivo small noncoding RNA:target RNA interactions by CIMERA-seq.

Small noncoding RNAs (sncRNAs) can regulate gene expression by guiding the RNA-induced silencing complex (RISC) to targeted transcripts for translational repression and/or target destabilization. Here, we present a robust benchtop protocol, termed CIMERA-seq, for the unambiguous profiling of sncRNA:target RNA interactions in a genome-wide and cell-type-selective manner. We describe steps for in vivo crosslinking and harvesting tissue, immunoprecipitation and covalent ligation of sncRNAs to target RNAs within the RISC, and sequencing of the resulting chimeric sncRNA:target RNA interactions.

Computational Analysis Tutorial for Chimeric Small Noncoding RNA: Target RNA Sequencing Libraries.

An understanding of the in vivo gene regulatory interactions of small noncoding RNAs (sncRNAs), such as microRNAs (miRNAs), with their target RNAs has been advanced in recent years by biochemical approaches which use cross-linking followed by ligation to capture sncRNA:target RNA interactions through the formation of chimeric RNAs and subsequent sequencing libraries. While datasets from chimeric RNA sequencing provide genome-wide and substantially less ambiguous input than miRNA prediction software, distilling this data into meaningful and actionable information requires additional analyses and may dissuade investigators lacking a computational background. This report provides a tutorial to support entry-level computational biologists in installing and applying a recent open-source software tool: Small Chimeric RNA Analysis Pipeline (SCRAP).