GSE168485
Addition-elimination mechanism activated nucleotide transition sequencing for RNA dynamics profiling
The temporal information of intracellular transcripts is essential to understand their functional roles, yet routine RNA-seq methods only measure RNA species at a steady state. Here we develop addition-elimination mechanism activated nucleotide transition sequencing (AENT-seq) for transcriptome-wide profiling of RNA dynamics. In AENT-seq, intracellular transcripts are metabolically labeled with 4-thiouridine (4sU), and then isolated total RNA are subjected to 4sU-to-cytosine (C) transition reactions based on a mild addition-elimination chemistry. N2H4•H2O is demonstrated to convert 4sU to 4-hydrazino cytosine (C*) in RNA-friendly aqueous conditions. The C* is recorded as natural C during extension reactions with a high efficiency of ~95%. This 4sU-to-C transition marks 4sU-containing nascent transcripts, and it enables subsequent sequencing analysis of RNA dynamics with single-nucleotide resolution. We applies our AENT-seq to simply investigate transcript dynamic information of several genes involved in cancer progression and metastasis. This method avoids harsh chemical conditions and harmful reagents involved in previous approaches, allowing rapid dissemination and extensive applications of the technique. Overall design: Next generation sequencing (NGS) provided the numbers and locations of 4sU-to-C (T-to-C) transition in the mapping reads of mRNA transcripts