22G-RNAs distal to piRNA target sites require the nuclear RNAi pathway.

<p>A) Small RNA high-throughput sequencing reads with unique matches antisense to the <i>piRNA sensor</i> from wild type and various mutant animals as indicated. The values of the <i>y</i>-axes correspond to reads matching the <i>piRNA sensor</i> normalised to reads matching Histone 2B (<i>his-58</i>). The x-axes represent the relative position of reads in the <i>piRNA sensor</i> transgene with numbers representing nucleotides from the start codon (set as 0). The transgene structure is schematically represented at the bottom. Colour code: green = GFP, grey = Histone 2B (<i>his-58</i>), dark blue = 21UR-1 target site plus/minus 50 bp, light blue = <i>tbb-2</i> 3′UTR. B) Left panel: Enrichment of small RNA high-throughput sequencing reads with matches antisense to the <i>piRNA sensor</i> in HRDE-1 Immunoprecipitation (IP). Displayed is the fold enrichment of reads found in anti-HRDE-1 IP from wild type versus <i>hrde-1</i> mutant animals. X-axis, schematic representation of the transgene and colour code as in A). Right panel: Western blot of anti-HRDE-1 Immunoprecipitation from wild type (left 3 lanes) and <i>hrde-1</i> mutant (right 3 lanes) animals. Antibodies used for western blot are anti-HRDE-1 and anti-PRG-1 as loading control. Inp. = 1% input, sup. = supernatant, IP = Immuoprecipitate. On the left, the relative migration of the 130 and 100 kDa bands of the PAGE Ruler Plus (MBI Fermentas) marker are indicated.