Rna gel loading dye

We confirmed that RINs were over 9.0 in all samples using the Agilent 2100 Bioanalyzer microfluidics-based platform. Total RNA (10 µg) was labelled with [5′-³²P] pCp (cytidine 3′,5′-bis[phosphate]) (0.11 pmol/μL in a total reaction volume of 30 μL) (PerkinElmer; NEG019A) using T4 RNA ligase 1 (NEB, M0204S) at 16°C overnight. Labelled RNAs were incubated at 85°C for 5 min and placed on ice. Then, labelled RNAs were digested with Ribonuclease A (50 ng/μL, Sigma) and Ribonuclease T1 (1.25 U/μl, ThermoFisher Scientific) at 37°C for 2 h in digestion buffer (100 mM Tris-HCl [pH7.5], 3M NaCl, 0.5 μg/mL yeast tRNA). Reactions were stopped by adding 5x stop solution (10 mg/mL Proteinase K, 0.125 M EDTA, 2.5% SDS) and subsequently incubating at 37°C for 30 min. After adding 400 μL of RNA precipitation buffer (0.5 M NH₄OAc, 10 mM EDTA), digested RNA samples were purified by phenol-chloroform extraction and isopropanol precipitation. Final products (10 μL) were mixed with RNA Gel loading Dye (NEB, R0641) and incubated at 95°C for 2 min. Then, samples were fractionated on 8 M urea-10% polyacrylamide denaturing gels (0.8 mm thick). Markers (Prestained Markers for small RNA Plus, BioDynamics Laboratory DM253) were also loaded. The gel was analysed using a Typhoon FLA 9500 Fluorescence Imager (GE Healthcare). Band intensity was quantified using Image J.