Analysis of the Argonaute 4-associated non-coding RNA in Arabidopsis thaliana sheds novel insights into gene regulation through RNA-directed DNA methylation

RNA-directed DNA methylation (RdDM) is a de novo DNA methylation mechanism in plants that plays a fundamental role in plant defence against invasive DNA and in maintaining genome stability by silencing transposons and repetitive sequences. Using nuclear RNA immunoprecipitation, we constructed a highly enriched library and obtained sequences of ncRNAs specifically associated with Argonaute 4 (AGO4), a key component of RNA-directed DNA methylation. A negative immunoprecipation (IP) library was prepared using IP RNA from tissues that don't express the target protein. A nuclear RNA library was also prepared using nuclei isolated RNA. Three libraries (1X FLAGAGO4 IP, 1X negative IP, 1X nuclear RNA). RNA-IP or nuclear RNA seq cDNA libraries were prepared using the template-switch cDNA library preparation method derived from Zhao et al (2010), and subjected to Illumina GAIIx single end sequencing (100bp)