Typhoon fla 9500 fluorescence imager

The unnatural sugars or chemoenzymatically labelled samples were precipitated with methanol chloroform. The protein pellets were re‐dissolved in 50 μL PBS buffer containing 1% SDS (wt/vol); then, 50 μm CuSO₄‐BTTAA premixed complex (CuSO₄‐BTTAA, molar ratio 1 : 2), 100 μm alkyne‐probes and 12.5 mm fresh sodium ascorbate were added for click reaction at 25 °C for 2 h. The reaction product was used for in‐gel fluorescence scanning by Typhoon FLA 9500 Fluorescence Imager (GE Healthcare, Life Sciences) or precipitated with methanol chloroform to remove click reagents for further O‐GlcNAcylation site identification.

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.