Anti dgcr8

1 × 10⁷ mESCs were washed in 1× cold PBS, scraped, and then lysed with a buffer containing 0.5% Nonidet, 0.5 mM DTT, 20 mM Tris-HCL, pH 7.5, 150 mM KCl, 2 mM EDTA, and inhibitors of RNases, proteases, and phosphatases (Thermo Fisher Scientific). 10% of total lysate was removed and kept as the input samples and the remainder used for immunoprecipitation. 10 µg anti-DGCR8 (Proteintech) or anti-IgG (Sigma-Aldrich) antibody was bound to Sepharose beads (Protein A; Invitrogen) in the presence of heparin. Precleared lysates were then incubated with the appropriate antibody-bound beads, and the immunoprecipitated proteins were then washed (150 mM KCl, 25 mM Tris, pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP-40, and RNase, protease, and phosphatase inhibitors) and incubated with DNase I in the presence of DNase buffer (Promega) followed by protease K (New England Biolabs, Inc.) in the presence of 2× protease buffer (New England Biolabs, Inc.). RNA extraction was then performed using phenol-chloroform extraction and ethanol/sodium acetate precipitation. RNA pellets were washed in ethanol, resuspended in 100 µl water, and quantified using a BioPhotometer (Eppendorf).