Shrhino flies

Nuclear run-on/GRO-seq experiments were performed on ovaries of shRhino flies (Bloomington Stock Center, 3407) driven by Nos-Gal4 (Bloomington Stock Center, 4937). Nos-Gal4 flies were used as a control. The nuclear run-on procedure was carried out as previously described (Shpiz et al. 2011 (link)) with slight modifications. BrUTP (5′-bromouridine-5′-triphosphate; Sigma, B7166)-labeled NRO-RNA was filtered through Illustra MicroSpin G25 columns (27-5325-01) twice to remove unincorporated BrUTP. The NRO-RNA was captured using the anti-BrdU antibody (Sigma, 032M 4753) for 1 h followed by incubation with Protein G beads (Dynabeads, Invitrogen, 1003D) for 1 h. The immunoprecipitation procedure was sequentially performed three times to yield highly enriched BrUTP RNA. As a negative control, the same procedure was performed on nonlabeled, total Drosophila ovary RNA. As a quality control, RT-qPCR was performed on 10% of the purified RNA with primers for Vasa, Rp49, and selected piRNA clusters. Libraries were cloned with the NEBNext Ultra Directional RNA library kit (E7420S) and sequenced on the Illumina HiSeq 2000 (50-bp reads) platform. Reads were mapped uniquely to the dm3 genome using Bowtie 0.12.7 (Langmead et al. 2009 (link)), allowing for no mismatches. The number of reads from the respective clusters are shown in Supplemental Table 5.

Strains T1 and BX2 were described in Dorer and Henikoff (1997) (link) and de Vanssay et al. (2012) (link). The strain P-1152, which carries the insertion of the P{lArB} construct in telomeric sequences of the X chromosome (site 1A), was described in Roche and Rio (1998) (link). The strain BC69 that has the insertion of the P{A92} construct at a euchromatic location on chromosome 2L (site 35B10-35C1) was described in Lemaitre et al. (1993) (link). Both stocks were a generous gift from S. Ronsseray. The Rhino-GFP fly line (GFP-tagged Rhino driven by the rhino promoter) was a generous gift from W. Theurkauff. The Cutoff-EGFP fly line (Nanos-GAL4/UASp-Cutoff-GFP), cuff^wm25, and cuff^qq37 were a generous gift from T. Schupbach. The Rhino-BioTAP flies were made by fusing the BioTAP tag (Alekseyenko et al. 2014 (link)) to the C-terminal region of the rhino gene under the UASp promoter. shRhino flies were obtained from the Bloomington Stock Center (stocks 34071 and 35171) and driven by Nos-Gal4 (stock 4937).