Dnase solution

DE (calcined powder), acetic acid (99.7%), dimethyl suberimidate dihydrochloride (DMS, 98%), lysozyme solution (30 mg/mL in distilled water), (3-aminopropyl) triethoxysilane (APTES, 99%), 3-aminopropyl(diethoxy)methylsilane (APDMS, 97%), [3-(2-aminoethylamino) propyl] trimethoxysilane (AEAPTMS, 80%), and N1-(3-trimethoxysilylpropyl) diethylenetriamine (TPDA, technical grade) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Ethanol (99%) and phosphate-buffered saline (PBS, 10×, pH 7.4) were ordered from Thermo Fisher Scientific, Waltham, MA, USA. DNase solution (1500 Kunitz units RNase-free DNase I) and Proteinase K solution (>600 mAU/mL) were purchased from Qiagen, Germany. Brucella agar was purchased from MB cell, Seoul, Korea. 5% defibrinated sheep blood was purchased from Kisan Bio, Seoul, Korea. nutrient broth and trypticase soy broth were purchased from BD Difco, Sparks, MD, USA. Sabouraud dextrose agar with chloramphenicol was purchased from Hardy Diagnostics, Santa Maria, CA, USA. All the regents were used without any further purification.

Full protocol is described in Supplementary Methods. Briefly, lysates were generated from HEK293T cells expressing exogenous GSK3β mutants and Flag-Drosha and incubated with anti-Flag affinity gel (Sigma) overnight at 4°C with rotation. Beads were washed, reconstituted with DNase solution (Qiagen) and treated with proteinase K. RNA was then extracted from beads using Trizol LS (Life Technologies) according to manufacturer's instructions and qRT-PCR performed for pri-miRs.