Deep sequencing of uncapped RNA in rdr1/2/6 and wild-type Arabidopsis plants

Our previous experiment on biogenesis mechanisim of stress-inducible antisense RNAs showed that accumulation of antisense RNA in a drought-inducible gene, RD29A locus was decreased in rdr1/2/6 triple mutant. The RD29A antisense RNA did not have CAP structure and poly(A) tail. We applied deep sequencing of uncapped RNA using SOLiD to identify RDR1/2/6-mediated antisense RNAs in genome-wide. We identified 1) 975 RDR1/2/6-mediated antisense RNA loci on which no. of sequence tags is decreased in rdr1/2/6 and makes peak and 2) 1,613 RDR1/2/6-mediated antisense RNA loci on which no. of the tags is decreased in rdr1/2/6 and enriched on antisense strand of exons. They were grouped into 1,959 non-redundant loci. Overall design: Untreated and 2hr drought stress-treated 2 weeks-old rdr1/2/6 and wild-type Arabidopsis thaliana plants were used. To determine 5’-end of uncapped RNA with and without poly (A), we developed Deep Sequencing of Uncapped RNAs (DSUR) with reference to PARE analysis (German et al. 2009, Nature Protocol 4:356-362) and applied DSUR using SOLiD to identify genome-wide RDR1/2/6-mediated antisense RNA loci in Arabidopsis.