title: GSM4259804: High.MLE_+ATP [L56]; Drosophila melanogaster; RIP-Seq
library_construction_protocol: vitRIP was performed with highly purified recombinant protein and 2 µg of total RNA, extracted from Drosophila cell lines or Drosophila heads. Two microgram of total RNA (10% was kept as RNA input) was incubated with purified recombinant protein for 30 minutes at 25°C in a total volume of 100 µl in vitRIP-100 buffer (25 mM HEPES-NaOH pH 7.6, 100 mM NaCl, 0.05% NP40, 3 mM MgCl2) supplemented with 10 µg BSA, 0.1 U/µl RNase-free recombinant DNase I and 0.8 U/µl RNasin recombinant RNase inhibitor with or without 1 mM ATP. Protein-RNA complexes were retrieved in 45 minutes at room temperature (22°C) on a rotating wheel using anti-FLAG, anti-MLE and anti-MSL1 antibodies, respectively. Following three washing steps, RNA was extracted using Proteinase K, phenol-chloroform extraction and ethanol precipitation in presence of 20 µg glycogen. Input material (10%) was treated equally. RNA-seq libraries were prepared with 30-40 ng of RNA material. Native RIP of endogenous MLE (eMLE) was essentially performed as described in (Ankush Jagtap et al., 2019) with modifications. For each replicate of specific (anti-MLE) and mock (bead control) RIP, 1 x 10e8 exponentially grown S2 cells were suspended in 1 ml of cold lysis buffer (20 mM HEPES-NaOH pH 7.6, 125 mM NaCl, 0.05 % SDS, 0.25 % sodium deoxycholate, 0.5 % NP40, 1.5 mM MgCl2, 0.25 mM DTT) supplemented with 0.05 U/µl RNase-free recombinant DNase I, 0.4 U/µl RNasin recombinant RNase inhibitor and 1x Complete EDTA-free protease inhibitor. Following incubation for 15 minutes on ice, the lysate was cleared by centrifugation and incubated with mock or specific anti-MLE antibody at 4°C for 2 hours on a rotating wheel. Following three washing steps, RNA was extracted using Proteinase K, phenol-chloroform extraction and ethanol precipitation in presence of 20 µg glycogen. Input material (2.5% of lysate) was treated equally. Of each sample, 40 ng RNA was used to prepare rRNA-depleted RNA-seq libraries. Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat; New England Biolabs) and samples were analyzed on a Bioanalyzer using the RNA 6000 Pico Kit (Agilent). Of the rRNA-depleted samples directional libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) following the recommended protocol. The quality of the libraries was assessed on a Bioanalyzer using the DNA 1000 or DNA High Sensitivity Kit (Agilent). Libraries were sequenced on an Illumina HiSeq1500 instrument in paired-end mode.
attributes.tag: GEO Accession
attributes.value: GSM4259804