Ab36764

Mouse sorted germ cells were homogenized with lysis buffer (20 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 200 mM NaCl, 0.05% (v/v) NP-40, 0.1 mM EDTA, 1 mM 4-(2- Aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.3 µM Aprotinin, 40 µM Bestatin, 10 µM E-64, 10 µM Leupeptin) and then centrifuged at 20,000 × g for 20 min at 4°C, retaining the supernatant. Anti-MIWI (Wako, Cat# 017–23451, RRID:AB_2721829, ~5 µg) or anti-MILI (Abcam Cat# ab36764, RRID:AB_777284, ~5 µg) antibodies were incubated with rotation with 30 µl of Protein G Dynabeads (Thermo Fisher, 10003D) in 1× PBS containing 0.02% (v/v) Tween 20 (PBST) at 4°C for 1 h. The bead-antibody complex was washed with PBST. Freshly prepared testis or cell lysate were added to the bead-antibody complex and incubated with rotation at 4°C overnight. The next day, the beads were wash ed once with lysis buffer and three times with 0.1 M Trisodium Citrate. After washing, RNA was purified with Trizol reagent (Thermo Fisher, 15596026) and used for small RNA library preparation. Each experiment was conducted for two independent biological replicates. The specificity of the commercial anti-MILI antibody was confirmed by immunoprecipitation from lysate of Mili^−/− whole testis (data not shown).