A Quantitative Sequencing Method for 5‐Formylcytosine in RNA.

5‐Formylcytosine (f5C) modification is present in human mitochondrial methionine tRNA (mt‐tRNAMet) and cytosolic leucine tRNA (ct‐tRNALeu), with their formation mediated by NSUN3 and ALKBH1. f5C has also been detected in yeast mRNA and human tRNA, but its transcriptome‐wide distribution in mammals has not been studied. Here we report f5C‐seq, a quantitative sequencing method to map f5C transcriptome‐wide in HeLa and mouse embryonic stem cells (mESCs). We show that f5C in RNA can be reduced to dihydrouracil (DHU) by pic‐borane, and DHU can be exclusively read as T during reverse transcription (RT) reaction, allowing the detection and quantification of f5C sites by a unique C‐to‐T mutation signature. We validated f5C‐seq by identifying and quantifying the two known f5C sites in tRNA, in which the f5C modification fractions dropped significantly in ALKBH1‐depleted cells. By applying f5C‐seq to chromatin‐associated RNA (caRNA), we identified several highly modified f5C sites in HeLa and mouse embryonic stem cells (mESC). [ABSTRACT FROM AUTHOR]