GSM3133728: Nup358GLKD GROSeq Biological Repeat 2; Drosophila melanogaster; OTHER
GRO-Seq was performed as described before (Core et al., 2008; Rozhkov et al., 2013) with the following changes. RNA fragmentation was performed using NEBNext Magnesium RNA Fragmentation Module as per manufacture instructions. Polynucleotide Kinase (PNK) treatment of RNA fragments was performed as described earlier (Gardini, 2017). Briefly, 1μl of RNase Inhibitor, Murine (NEB), 30 U of T4 PNK (NEB), 1mM ATP (ThermoFisher Scientific) and 1X PNK reaction buffer was added to purified RNA fragments and reaction was incubated at 37°C for 60 min. To this reaction, 20U of PNK and 10 mM EDTA were added and incubated at 37°C for an additional 60 min. Enzyme was heat inactivated by incubating the reaction mixture at 65°C for 20 min. Libraries were prepared using TruSeq small RNA Library Prep Kit (Illumina) and sequenced using single-end 50bp sequencing. GRO-seq was performed twice with two independent biological repeats of control and Nup358 GLKD ovaries.