Amersham hybond nx

Total cellular RNA was extracted from 1 × 10⁶ mESC pellets using TriZOL Reagent (Thermo Fisher Scientific). A total of 10 µg total RNA was resuspended in 30 µl final of 50% deionized formamide, loaded on a 17.5% acrylamide gel (30% acrylamide/bis solution 19:1; Bio-Rad Laboratories), blotted for 1 h on a nylon membrane (Amersham Hybond-NX; GE Healthcare) in 0.5× TBE (Tris/borate/EDTA) buffer at 25 V and 1.5 mA per square centimeter of membrane in a semidry system. Membranes were then ethyl-dimethyl-aminopropylcarbodiimide cross-linked. Prehybridizations and hybridizations were both performed in PerfectHyb Plus Hybridization Buffer (Sigma-Aldrich) at 42°C. All washes were performed in SSC 2×, SDS 0.1%. Radioactive signals were detected with an FLA-7000 device (Fujifilm). For subsequent reprobing, membranes were stripped with boiling 0.1% SDS. miRNA and U6 probes were generated by labeling specific oligonucleotides at the 5′ end using T4 polynucleotide kinase (New England Biolabs, Inc.) and 25 µCi γ[³²P]-ATP (3,000 Ci/mmol) and following the manufacturer’s instructions. Probes were then purified on Illustra MicroSpin G-25 Columns (GE Healthcare). All of the probes used for miRNA Northern blots are described in Table S1.

RNAs were resolved on a 8% urea-acrylamide gel, transferred on a nylon membrane (Amersham Hybond-NX, GE-Healthcare Life Sciences), crosslinked to the membrane by chemical treatment at 60°C using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (Sigma) for 1 h 30 min. MiRNAs and pre-miRNAs were detected with specific 5′-³²P labeled oligonucleotides (Supplementary Table S1). The signals were quantified using a Fuji Bioimager FLA5100. miR-16 was probed as a loading control.