Fig. 4

Phased siRNAs and highly expressed small RNAs of unknown function. a Exemplary genetic locus of a Kangaroo element harboring predicted phased siRNA loci. Upper panel: all reads measured in the VcAGO3 IP library, middle panel: read coverage of the predicted phased siRNA loci, lower panel: bar depiction of phased siRNA loci (black bars) and Kangaroo (blue). b Coverage of small RNAs associated with VcAGO3 of the consensus sequence (Repbase19.02) of the repetitive element Kangaroo. Red bars represent reads mapping to the plus strand, blue bars represent reads mapping to the negative strand. c Validation of expression of small RNA originating from a predicted phased RNA locus. d All reads of the putative intergenic fraction that are associated with VcAGO3 are displayed. The x-axis of the diagram represents the length of an artificial genome sequence that was created by aligning all available genome scaffolds in a row. This sequence was divided into 50 kb long sections and all reads that were mapped falling into one section were summed up and displayed by blue bars. The location for an exceptionally high read count was marked next to the corresponding bar. The number below each scaffold indicates the starting point of that section within the scaffold. Lower panel: blow-up of the section in scaffold 12 with the highest read count. e Pie chart visualizing the amount of small RNAs reads stemming from highly expressed hotspots from d with respect to all reads. f Validation of expression by RNA blot. The small RNA with the highest read count within the respective scaffold section (see d) was blotted. g Total RNA isolated from C. reinhardtii (Cre) or V. carteri (Vca) was blotted and robed for the indicated small RNAs. sRNA sequences of C. reinhardtii were taken from Molnar et al. [14]