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m 6 A-SAC-seq for quantitative whole transcriptome m 6 A profiling.

Ruiqi GeChang YeYong PengQing DaiYutao ZhaoShun LiuPingluan WangLulu HuChuan He
Published in: Nature protocols (2022)
N 6 -methyladenosine (m 6 A) is the most abundant mRNA modification in mammalian cells, regulating many physiological processes. Here we describe a method for base-resolution, quantitative m 6 A sequencing in the whole transcriptome. The enzyme and small-molecule cofactor used in this protocol are prepared by recombinant protein expression and organic synthesis, respectively. Then the library can be prepared from various types of RNA samples using a ligation-based strategy, with m 6 A modifications being labeled by the enzyme and cofactor. Detailed instructions on ensuing data analysis are also included in this protocol. The method generates highly reproducible results, uncovering 31,233-129,263 sites using as little as 2 ng of poly A + RNA. These identified sites correspond well with previous m 6 A profiling results, covering over 65% of peaks detected by the antibody-based approaches. Compared with other currently available methods, this method can be applied to various types of biological samples, including fresh and frozen tissues as well as formalin-fixed paraffin-embedded samples, providing a quantitative method to uncover new insights into m 6 A biology. The protocol requires basic expertise in molecular biology, recombinant protein expression and organic synthesis. The whole protocol can be done in 15 days, with the library preparation taking 5 days.
Keyphrases
  • single cell
  • randomized controlled trial
  • rna seq
  • data analysis
  • small molecule
  • gene expression
  • genome wide
  • high resolution
  • mass spectrometry
  • computed tomography
  • cell free
  • high throughput sequencing