Ovaries were dissected from adult flies and RNA was isolated with Ribozol Reagent. For standard non-polyA selected libraries, rRNA was depleted with Ribo-Zero™ rRNA Removal Kit. For polyA-selected libraries, selection was performed with the Dynabeads® mRNA Purification Kit. DNA for ChIP seq was prepared as in Le Thomas et al., 2014, using anti-RNA Pol II antibody from Abcam (ab5408). Nuclear run-on was performed as previously described (Shpiz et al.) with slight modifications. 5’-Bromouridine-5’-triphosphate (BrUTP; Sigma, B7166) labeled RNA was filtered through Illustra MicroSpin G25 columns (27-5325-01) twice to remove unincorporated BrUTP. The nuclear run-on RNA was captured using anti-BrdU antibody (Sigma, 032M 4753) by incubation for 1 hour followed by incubation with Protein G beads (Dynabeads, Invitrogen, 1003D) for 1 hour. Standard RNA-seq libraries were generated using the NEBNext Ultra Directional RNA Library Prep kit (NEB #E7420) according to the manufacturer's instructions. For global nuclear run-on (GRO-seq) (Core et al., 2008) immunoprecipitated RNA was used to clone total RNA libraries using the NEBNext Ultra Directional RNA Library Prep Kit (NEB #E7420). For small RNA librarires, 19-29 nt RNAs were isolated from total RNA of dissected ovaries using 12% polyacrylamide gels as described in previous studies (Aravin et al., 2008; Brennecke et al., 2007) and libraries were constructed using the Illumina TruSeq smallRNA Library Kit. ChIP-seq libraries were prepared using the NEBNext ChIP-Seq Library Prep Master Mix Set (#E6240).