Storage phospho screen

Relative levels of internal m⁶A was determined by thin layer chromatography (TLC) as described previously³⁵ . m⁶A measured using TLC does not have the problem of potential contamination by ubiquitous ribosomal RNA m⁶A and snRNA m⁶A since these m⁶A sites are found in a consensus site that prevents its detection by TLC³⁵ . 100 ng of poly(A) purified RNA was digested with 2U ribonuclease T1 for 2h at 37°C in the presence of RNAseOUT (Invitrogen). Digested RNA was 5′ labeled with 0.4 mBq [γ-³²P] ATP for 30 min at 37°C followed by removal of the γ– phosphate of ATP by incubation with 10U apyrase (NEB) for 30 min at 30°C. RNA was purified by phenol-chloroform extraction and ethanol precipitation and resuspended in 10 μl of DEPC-H₂O and digested to single nucleotides with 2U of P1 nuclease for 3h at 37°C. 1 μl of released 5′ monophosphates were analyzed by on glass-backed PEI-cellulose plates (MerckMillipore) as described previously^{36 (link)}. Plates were exposed to a storage phospho screen (GE Healthcare Life Sciences) below saturation and processed on a Typhoon Trio imager (GE Healthcare Life Sciences) at 200 μm resolution. Quantification of individual nucleotides was done with ImageJ (1.49v). The relative amount of m⁶A was calculated as a percent of the total of A, C, and U spots, as described previously³⁵ .