m⁶A-SAC-seq

A Method for Assaying m⁶A Epigenetic Modifications at Single Base Resolution

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Table of Content

Introduction

Run-the-pipeline

Dissect-the-pipeline

m⁶A selective allyl chemical labeling and sequencing (m⁶A-SAC-seq) for base-resolution m⁶A analysis

Strategy — The very similar chemical feature of m⁶A and A makes it challenging to differentiate the methylated versus the unmodified base in RNA. For instance, unlike modifications such as m¹A, m³C, m¹G and m²₂G, which occur at the Watson–Crick face of the nucleobases and could induce mutations or stops upon reverse transcription (RT), m⁶A shows a minor effect on Watson-Crick base pairing. The modification information will be lost upon RT followed by amplification. The inert chemical property of the methyl group on m⁶A so far precludes chemistry-based selective labeling of m⁶A versus A. We thus envisioned selective enzymatic reactions that could preferentially label or modulate either m⁶A or A. Ideally, our reaction preferentially labels m⁶A instead of A because A is ~200-fold more abundant than m⁶A in mammalian mRNA. A reaction that specifically modulates A over m⁶A would need to be extremely efficient and selective in order to quantitatively detect 0.4-0.6% of m⁶A in RNA. Based on these considerations we have invented m⁶A Selective Allyl Chemical labeling and sequencing (m⁶A-SAC-seq), a quantitative method for whole-transcriptome mapping of m⁶A at single-nucleotide resolution.