GSM9533293: shWhite_Piwi-IP; Drosophila melanogaster; ncRNA-Seq
Ovaries (600 µl packed volume per genotype) were dissected in ice-cold PBS and lysed in 1× RIPA buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM EDTA, 0.1 mM Pefabloc). Homogenates were cleared by centrifugation and diluted with 3 ml IP dilution buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl). Antibodies were coupled to M280 sheep anti-mouse IgG Dynabeads (Life Technologies). For Piwi and Aub immunoprecipitations, 150 µl antibody-coupled beads were incubated with 1.5 ml lysate; for Ago3, 300 µl beads were incubated with 3 ml lysate. Lysates were rotated at 4 °C overnight. Beads were collected and washed seven times with IP wash buffer (50 mM Tris HCl pH 7.5, 500 mM NaCl, 2 mM MgCl₂, 10% glycerol, 1% Empigen); for Piwi IPs, 150 mM NaCl was used. Bound RNA was isolated by acid phenol–chloroform extraction and ethanol precipitation. For visualization during cloning, 10% of the extracted RNA was labeled with [γ-³²P]-ATP. The recovered IP-derived small RNAs were used directly as input for the gel-based library preparation pipeline described above. Small RNAs were isolated by resolving the mixture on a 12.5% polyacrylamide–urea gel and excising the region flanked by the spike-in signals. These spike-ins were ligated to conventional 4N adapters alongside the endogenous small RNA population. Following each ligation step, libraries were gel-purified, with band excision guided by the radioactive signal. After reverse transcription and PCR amplification, the spike-in sequences were removed from the library via restriction digestion. The libraries  were pooled at the final stage using PCR-introduced dual or single index barcodes.