Analysis of Piwi-Loaded Small RNAs in Terahymena

Abstract

Scan RNAs (scnRNAs) are developmentally regulated siRNAs of ~26-32 nucleotides in length that are involved in programmed DNA elimination in Tetrahymena. scnRNAs are loaded onto the Piwi-related protein Twi1p and 2'-O-methylated at their 3' termini. We describe two alternative strategies for analyzing the Twi1p-loaded scnRNAs: preparation of loaded scnRNAs by immuno-purification of the Twi1p-scnRNA complex and exclusion of non-methylated scnRNAs during cDNA library construction using periodate oxidation.

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

“…Construction of small RNA cDNA libraries, high-throughput sequencing, and data processing were performed as previously described (Mochizuki and Kurth, 2013; Noto et al, 2014; Schoeberl et al, 2012). The data for Twi1p- and Twi11p-bound small RNAs have been deposited at the NCBI Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) as GSE79849 and GSM1672144, respectively.…”

A Tetrahymena Hsp90 co‐chaperone promotes si RNA loading by ATP ‐dependent and ATP ‐independent mechanisms

“…Total and Twi1p-bound scnRNAs were analyzed as described previously (Noto et al , 2010). High-throughput sequencing of small RNAs was performed as described previously (Schoeberl et al , 2012; Noto et al , 2014), except that 16- and 32-nt RNAs were used as size markers for the gel extraction steps. The raw sequencing data and processed data sets have been deposited at the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) as GSE61609.…”