3-methylcytidine modification status was explored through the PHA assay. To conduct the PHA assay, probes were designed to hybridize upstream and downstream of residue 32 (oligos listed in Supplementary Table 1). 5 μg of RNA was loaded onto a 10% polyacrylamide, 1xTBE, 7 M urea gel and transferred onto an Amersham Hybond-XL membrane (GE Healthcare) for Northern Blotting analysis. Oligonucleotides used to detect RNAs are listed in Supplementary Table 1. The oligos were radiolabeled by T4 polynucleotide kinase (NEB) with adenosine [γ32P]-triphosphate (6000 Ci/mmol, Amersham Biosciences) following standard procedures. Northern blots were visualized by Phosphor-Imager analysis and stripped via two incubations at 80 °C for 30 min in a buffer containing 0.15 M NaCl, 0.015 m Na-citrate and 0.1% SDS. Image analysis of Phosphorimager scans were performed using ImageJ open source software.
