T4 pnk buffer

Approximately 40 µg of total RNA was prepared for Northern blotting analysis of known and novel miRNAs. The samples used for qRT-PCR assays were pooled for RNA extraction from other three independent experiments with at least three biological replicates each. The total RNA was separated in a 15% urea polyacrylamide gel, electrophoretically transferred to Hybond-NX membrane (GE Healthcare, Buckinghamshire, UK) using a semi-dry transfer apparatus (Amersham Biosciences, Piscataway, NJ, USA), and was chemically cross-linked via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). For labeling reaction of probes, 1 µL of 10 µM probes, 2.5 µL of 10 × T4 PNK buffer (New England Biolabs, Beverly, MA, USA), 1 µL of [γ-³²P] ATP (~10 µCi/µL), 19.5 µL of ddH₂O, and 1 µL of T4 Poly Nucleotide Kinase (New England Biolabs, Beverly, MA, USA) were added (a total volume of 25 µL reaction) and kept in a water bath for 1 h at 37 °C. Blots were pre-hybridized and hybridized at 42 °C overnight using ULTRAhyb^®-Oligo hybridization buffer (Sigma-Aldrich, St Louis, MO, USA). Post-hybridization washes were performed using 2× SSC and 0.2% sodium dodecyl sulfate (SDS) at 42 °C for 20 min for twice. Hybridization signals were detected by exposing blots to autoradiographic film. The sequences of probes used for Northern blotting analysis were listed (Table S3).

Standard 5′ radiolabeling was performed with T4 polynucleotide kinase (New England Biolabs) at 0.2 U/μL (manufacturer’s units), 1X T4 PNK buffer (New England Biolabs), 400 nM DNA oligonucleotide, and 200 nM [γ−³²P]-ATP (PerkinElmer) for 30 min at 37°C, followed by a 20-min heat-killing incubation at 65°C. Radiolabeled oligos were then buffer exchanged into water using a Microspin G-25 spin column (GE Healthcare). For 5′ radiolabeling of sgRNAs, the 5′ triphosphate was first removed by treatment with Quick CIP (New England BioLabs, manufacturer’s instructions). The reaction was then supplemented with 5 mM DTT and the same concentrations of T4 polynucleotide kinase (New England BioLabs) and [γ−³²P]-ATP (PerkinElmer) used for DNA radiolabeling, and the remainder of the protocol was completed as for DNA.

T4 polynucleotide kinase (NEB) was used to end label oligomers using γ-³²P ATP in T4 PNK buffer (NEB) as described before^{38 (link)}. Reaction was incubated for 1 h at 37 °C and then stopped using 10 mM Tris-1 mM EDTA buffer. Labelled substrates were purified using Sephadex G-25 column and then stored at −20 °C until use. Annealing of complementary oligomers was done by boiling oligomers for 10 minutes in the presence of 100 mM NaCl and 1 mM EDTA, in 1:5 ratio (labelled: unlabelled)^{37 (link),38 (link)}.