Endogenous small RNA Analysis Protocol

For endogenous sRNA analysis, small RNA samples were extracted from mycelia grown 48 h on YPG plates using the miRVana kit (Ambion), following the instructions of the supplier. cDNA libraries of small RNAs were generated and sequenced as described previously [11 (link)]. For isolation of sRNAs bound to the Ago-1 protein, small RNA samples were extracted from Ago-1-containing fractions and used to construct the cDNA library as previously described [11 (link)]. Equivalent fractions from the ago-1^- mutant were used for isolation of sRNAs as a negative control. Sequencing of the Ago-1 bound cDNA libraries were carried out as described [14 (link)]. For detection of endogenous sRNAs in Northern blot experiments, membranes were hybridized as described [22 (link)] with sense and antisense-specific riboprobes prepared by in vitro transcription (MAXIscript transcription kit; Ambion) of linearized plasmids containing specific sequences for each locus (S2 Table).