Analysis of Piwi-Loaded Small RNAs in Terahymena

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.