A low-cost, low-input method establishment for m6A MeRIP-seq

Abstract

Abstract N6-methyladenosine (m6A) is a highly prevalent modification found in mammal mRNA molecules that plays a crucial role in the regulation of cellular function. m6A RNA immunoprecipitation sequencing (MeRIP-seq) has been frequently used in transcriptomics research to identify the location of m6A. MABE572 (Millipore) is the most widely utilized and efficient anti-m6A antibody for MeRIP-seq. However, due to the high dose and price of this antibody, which has also been taken off the market, we discovered that CST's anti-m6A antibody can be used instead of MABE572 to map the m6A transcriptome. In the present study, we performed different concentrations of the CST anti-m6A antibodies with the corresponding initiation RNA of HEK293T cells, 2.5 μg antibody with 1 μg total RNA, 1.25 μg antibody with 0.5 μg total RNA, and 1.25 μg antibody with 0.1 μg total RNA. By comparing the m6A peak calling, enriched motifs, alternative splicing events, and nuclear transcripts modified by m6A between the CST and Millipore libraries, it was found that the CST library presented similar data to Millipore, even at incredibly low doses. The volume and cost of antibodies are significantly reduced by this refined MeRIP-seq using CST antibody, making it convenient to map future large-scale sample m6A methylation.