GRO.AubWT
GRO-seq was performed as described with slight modifications (Rozhkov et al., 2013) and sequenced using a NextSeq 500 (Illumina). Briefly, one-to-two-day-old female flies were fed in yeast for three days before their ovaries were dissected. One hundred pairs of ovaries were homogenized in HB35 buffer (15 mM HEPES-KOH, pH 7.5, 10 mM KCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 0.05% [v/v] NP-40, 0.35 M sucrose, 1 mM dithiothreitol, 1 mM 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, 0.3 µM Aprotinin, 40 µM Bestatin, 10 µM E-64, 10 µM Leupeptin). Nuclei were purified by passing twice through sucrose cushions that contain 800 µL HB80 buffer (15 mM HEPES-KOH, pH 7.5, 10 mM KCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 0.05% [v/v] NP-40, 0.80 M sucrose, 1 mM dithiothreitol, 1 mM 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, 0.3 µM Aprotinin, 40 µM Bestatin, 10 µM E-64, 10 µM Leupeptin) on the bottom phase and 350 µL HB35 buffer on the top. Nuclei were washed once with 500 µL freezing buffer (50 mM Tris-HCl, pH 8.0, 40% [v/v] glycerol, 5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, 0.3 µM Aprotinin, 40 µM Bestatin, 10 µM E-64, 10 µM Leupeptin) and frozen in liquid nitrogen with 100 µL freezing buffer. To carry out nuclear run-on assay, 100 µL freshly prepared reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 300 mM KCl, 1% [w/v] sarkosyl, 500 µM ATP, 500 µM GTP, 500 µM Br-UTP, 2.3 µM CTP, 1 mM dithiothreitol, 20 U Promega RNasin Plus RNase Inhibitor) was added to nuclei and incubated at 30°C for 5 min. RNA was extracted using Trizol (Invitrogen). Nascent RNAs with Br-UTP incorporated were enriched by immunoprecipitation using anti-5-bromo-2′-deoxyuridine antibody (clone PRB-1; EMD Millipore, Billerica, MA) as described (Shpiz and Kalmykova, 2014), followed by rRNA depletion using RNase H (Adiconis et al., 2013), fragmentation, and library construction (Zhang et al., 2012).