Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA
Qing Dai, Chang Ye, Iryna Irkliyenko, Yiding Wang, Hui‐Lung Sun, Yun Gao, Yushuai Liu, Alana V. Beadell, José Perea, Ajay Goel, Chuan He
Abstract
Abstract Bisulfite sequencing (BS-seq) to detect 5-methylcytosine (5mC) is limited by lengthy reaction times, severe DNA damage, overestimation of 5mC level and incomplete C-to-U conversion of certain DNA sequences. We present ultrafast BS-seq (UBS-seq), which uses highly concentrated bisulfite reagents and high reaction temperatures to accelerate the bisulfite reaction by ~13-fold, resulting in reduced DNA damage and lower background noise. UBS-seq allows library construction from small amounts of purified genomic DNA, such as from cell-free DNA or directly from 1 to 100 mouse embryonic stem cells, with less overestimation of 5mC level and higher genome coverage than conventional BS-seq. Additionally, UBS-seq quantitatively maps RNA 5-methylcytosine (m 5 C) from low inputs of mRNA and allows the detection of m 5 C stoichiometry in highly structured RNA sequences. Our UBS-seq results identify NSUN2 as the major ‘writer’ protein responsible for the deposition of ~90% of m 5 C sites in HeLa mRNA and reveal enriched m 5 C sites in 5′-regions of mammalian mRNA, which may have functional roles in mRNA translation regulation.