Nos gal4

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.

All flies were fed with standard corn meal food at 25°C. Information about the alleles and transgenes used in the present study are available either in FlyBase (www.flybase.org) or as stated: Nos-Gal4 (4937; Bloomington Drosophila Stock Center, Dept Biology, Indiana University, Bloomington, IN, USA), bam-Gal4; Δ86/+ was a gift from Professor Dahua Chen (Institute of Zoology, Chinese Academy of Sciences, Beijing, China) and has been described in a previous study (11 (link)). All UAS-RNAi transgenic fly lines were obtained from The TsingHua Fly Center (THFC; Beijing, China). Drosophila melanogaster Canton-Special flies were used as the wild type (WT) strain.

All fly stocks were raised in standard Bloomington medium at 25°C. The following fly stocks were used: nos-gal4 (Van Doren et al., 1998 (link)), cnn^HK21 (Megraw et al., 2001 (link)), and UAS-dpp (obtained from the Bloomington Drosophila Stock Center, Bloomington, IN); Ubi-Pavarotti-GFP and UAS-Pavarotti-GFP (Minestrini et al., 2002 (link); obtained from David Glover, University of Cambridge); cnn^mfs3 (Megraw et al., 1999 (link); obtained from Thom Kaufman, Indiana University); dsas-4^S2214 (Basto et al., 2006 (link); obtained from the Bloomington Stock Center); and UAS-Upd (Zeidler et al., 1999 (link)), Asl(asterless)-YFP (Varmark et al., 2007 (link)), and Ubi-Cnb-YFP (Januschke et al., 2011 (link); obtained from Cayetano Gonzalez, IRB Barcelona). To assess MR inheritance in cnn mutants, a cnn^HK21/CyO; Ubi-PavGFP/TM3 fly stock was generated, which was subsequently crossed with cnn^mfs3/CyO to obtain transheterozygous mutant flies (cnn^HK21/cnn^mfs3; Ubi-Pav-GFP/+), as well as control siblings (cnn/CyO; Ubi-Pav-GFP/+).