title: GSM8770577: Siwi-loaded piRNA, rep2; Bombyx mori; ncRNA-Seq
library_construction_protocol: Dynabeads Protein G (Thermo Fisher Scientific) and antibodies were incubated with 0.02% Tween-20 in PBS. Anti-Siwi and anti-AGO3 (Nishida et al. 2015) antibodies were used. The 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] supplemented with RNasin Plus Ribonuclease Inhibitor (Promega). After the cell debris was removed by centrifugation, the cell lysates were incubated with antibody-conjugated beads at 4°C. After protein-antibody binding, the beads were washed once with IP buffer containing 500 mM NaCl and then extensively washed with IP buffer. Protein-bound RNAs were purified from the beads using proteinase K (Roche) treatment followed by phenol/chloroform treatment, and precipitated with ethanol. Small RNA libraries were prepared using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs) and sequenced on the MiSeq platform (Illumina) to obtain 51 nt single-end reads.
identifiers.namespace: GEO
identifiers.id: GSM8770577_r1