Isolation and profiling of protein-associated small RNAs

Small RNAs are short noncoding RNAs with important regulatory roles in many cellular processes. Small RNAs are generated by DICER or DICER-like proteins and then incorporated into RNAi effector -proteins ARGONAUTEs (AGOs) for silencing of their targets. In plants, small RNAs regulate host innate immunity against various pathogens, but their mode of action and associated protein factors that facilitate their function remain to be elucidated. Here, we describe an efficient method to isolate -AGO-associated small RNAs from Arabidopsis. This protocol can be easily adapted for the isolation of any protein-associated small RNAs. We utilized immunoprecipitation tandem with deep sequencing to identify small RNAs with functions in plant innate immunity. Using this described protocol, we identified miR393* that plays a crucial role in plant antibacterial defense. The distinct roles played by individual AGO proteins were observed.