Analysis of Schizosaccharomyces pombe Heterochromatin-Associated Short Interfering RNAs.

An important aspect of RNAi-mediated heterochromatin assembly is the production of short interfering RNAs (siRNAs) in cis at the heterochromatic target. These populations of small RNAs (20-30 nt) can be detected via northern blot analysis with radiolabeled RNA probes corresponding to the siRNAs. Most heterochromatin-associated siRNAs originate from both strands of the pericentromeric dg and dh repeats. Thus, production of RNA probes by in vitro transcription utilizes a DNA template with sequences corresponding to either dg or dh repeats. Alternatively, radiolabeled DNA probes, which are somewhat easier to prepare, may be used. A control probe against a small RNA, such as a tRNA, may also be prepared for loading. More recently genome-scale analysis using next-generation sequencing platforms has enabled the detection of rare siRNAs at sites other than the major heterochromatin domains. This protocol describes procedures for the extraction of small RNAs from Schizosaccharomyces pombe and their detection by northern blot analysis.