Instrument: Illumina MiSeq
Strategy: RIP-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Dynabeads Protein G (Invitrogen) and anti-Flag antibodies (Sigma, M2 mouse monoclonal F3165) were incubated in T-PBS containing 0.02% Tween at room temperature. BmN4 cells were lysed at 4°C with immunoprecipitation (IP) buffer [30 mM HEPES-KOH (pH 7.3), 150 mM KOAc, 2 mM Mg(OAc)2, 5 mM DTT, 0.1% NP-40, 2 μg/mL pepstatin, 2 μg/mL leupeptin, and 0.5% aprotinin] containing RNasin Plus Ribonuclease Inhibitor (Promega). After the cell debris was removed by centrifugation, the cell lysates were incubated with antibodies-conjugated beads for 1 h at 4°C. After protein-antibody binding, the beads were washed once with IP buffer containing 500 mM NaCl and then extensively with IP buffer. The protein-bound RNAs were purified from the beads by proteinase K (Roche) treatment, phenol/CHCl3 treatment, and precipitated with ethanol. RNAs of 22–35 nt in length were separated on denaturing (6 M urea) PAGE and used to generate RNA libraries. PIWI-associated small RNA libraries were prepared from Luc, Mael, and Ago3 KD BmN4 cells with the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs) and sequenced using Illumina MiSeq to obtain 51 nt single reads.