γ 32p atp

The RNA of four repetitive RRACH sequences 5′-GGACUGGACUGGACUGGACU-3′ (Wang et al. 2016b (link)) used also in the m⁶A methylation assay was labeled with radioactive phosphate. One micromole of RNA oligonucleotides were incubated with 20 µCi of [γ-³²P]ATP (Hartmann Analytic) and 0.5 U/µL T4 Polynucleotide Kinase (PNK) in 1× PNK buffer A (Thermo Scientific) at 37°C for 30–60 min. The labeling reaction was stopped by EDTA, pH 8.0 and purified afterwards using MicroSpin G-25 columns (GE Healthcare Life Sciences) according to the manufacturer's protocol.

All substrates were buffer exchanged into 50 mM HEPES pH 7.5, 50 mM NaCl, 0.5 mM TCEP, 10% glycerol. Kinase reactions with yeast Cdk1-Clb2 were performed as previously described^{7 (link)}. Briefly, prior to adding substrates, ~1-5 nM CDK complexes and 200 nM Cks1 were incubated in the presence of 10 mM MgCl₂ and 100 μM ATP for 45 min. Phosphorylation reactions were initiated by combining 25 nCi [γ-³²P]-ATP (Hartmann Analytic) and 1-2 μg of substrate with the CDK-Cks1 mixture in kinase buffer (25 mM HEPES pH 7.5, 50 mM NaCl 10 mM MgCl₂, 1 mM DTT, 100 μM ATP) and incubated at 25°C. Reactions were arrested at specific time points by addition to SDS-PAGE sample buffer, and products were analyzed by Phos-tag SDS-PAGE with a gel composition of either 6 or 7.5% acrylamide and 50 μM Phos-tag (Wako Chemicals). Autoradiography was performed with an Amersham Typhoon 5 Biomolecular Imager (GE Healthcare Life Sciences), and images were quantified using ImageQuant TL software (Amersham Biosciences). Phosphorylation reactions with human kinase were performed similarly except with 100 nM final concentration of CDK.

The sequence for the let-7a standard used for northern blotting was the same as mentioned above but with 5′P. Northern blot probes were labeled by incubating RNA with 20 μCi of γ- ³²P-ATP (Hartmann Analytic) and 0.5 U/μl T4 Polynucleotide Kinase (PNK) in 1x PNK buffer A (Thermo Scientific) at 37°C for 30–60 min. The reaction was stopped by adding ethylenediaminetetraacetic acid, pH 8.0, and labeled oligonucleotides were purified with a G-25 column (GE Healthcare). Oocytes were collected in 1× PBS with Ribolock, RNA was isolated with Trizol and separated in 1× TBE in 12% polyacrylamide urea gel. The RNA was then blotted for 30 min at 20 V onto Amersham Hybond-N membrane (GE Healthcare) and crosslinked to the membrane for 1 h at 50°C using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution. The membrane was incubated overnight at 50°C in hybridization solution (5× SSC, 7% sodium dodecyl sulphate (SDS), 20 mM sodium phosphate buffer pH 7.2, 1× Denhardt's solution) with a ³²P-labeled oligonucleotide antisense to the small RNA. The membrane was washed twice with 5× SSC, 1% SDS, once with 1× SSC, 1% SDS and wrapped in Saran. Signals were detected by exposure to a storage phosphor screen and scanning with Personal Molecular Imager (Bio-Rad). For further details, refer to reference (40 (link)).