GSM9533223: CLIP_INPUT; Drosophila melanogaster; OTHER
Harvested cell pellets were lysed in lysis buffer (30 mM HEPES-KOH pH 7.4, 150 mM NaCl, 2 mM MgCl₂, 0.5% Triton X-100, 0.2 mM DTT, 0.2 mM Pefabloc) and clarified by centrifugation. Lysates were treated with RNase I (0.08 U per 100 µl cell pellet; Thermo Fisher) for 30 min at room temperature, followed by inhibition with SUPERase In (Thermo Fisher). Immunoprecipitation was performed using monoclonal anti-Yb antibodies coupled to Dynabeads M-280 sheep anti-mouse in TBS-Tx. Beads were incubated with lysates for 4 h at 4 °C and washed four times with high-salt wash buffer (30 mM HEPES-KOH pH 7.4, 1 M NaCl, 2 mM MgCl₂, 1% Empigen). On-bead RNA end repair was performed using T4 polynucleotide kinase (T4 PNK) without ATP to open 2′,3′-cyclic phosphates, followed by 5′-OH radiolabeling with [γ-³²P]-ATP and final phosphorylation with 10 mM cold ATP. Protein–RNA complexes were eluted in SDS buffer, resolved by 8% SDS-PAGE, and transferred to a 0.45 µm nitrocellulose membrane. Complexes were visualized by autoradiography, and corresponding membrane regions were excised and digested with proteinase K (1 mg/ml). RNA was extracted via acid phenol–chloroform, precipitated, and used for small RNA library preparation. Libraries were generated following standard gel based small RNA cloning and sequenced on an Illumina platform (single-end 50 bp).