Total RNA from dissected ovaries was purified using Trizol according to manufacturer's instructions. Chromatin-associated RNAs were isolated as described previously (Khodor et al., 2011), with modifications.  For total RNA-sequencing and input RNA in Thoc5-GFP RIP experiment, ribosomal RNA was depleted using the Ribo-Zero rRNA Removal Kit (human/mouse/rat, Epicenter) with additional Drosophila-specific probes. Thoc5-GFP and GFP-bound RNA (negative control) was isolated from immunopurified protein bound RNA fraction from ovarian lysates using antibodies against GFP. Detailed protocols for chromatin-associated RNAs and protein-bound RNA extraction is available in the accompanying manuscript. Total and chromatin-associated RNA libraries were generated using the NEBNext library preparation kit (NEB) according to the manufacturer's instructions. For small RNA librarires, 19-29 nt RNAs were isolated from total RNA of dissected ovaries using 12% polyacrylamide gels, and library construction was performed as described in previous studies (Aravin et al., 2008; Brennecke et al., 2007).
GEO Accession
GSM2091550