N6-Methyladenosine (m6A) accounts for approximately 0.2% to 0.6% of all
adenosine in mammalian
mRNA, representing the most abundant internal
mRNA modifications.
m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) is a powerful technique to map the
m6A location transcriptome-wide. However, this method typically requires 300 μg of total
RNA, which limits its application to patient
tumors. In this study, we present a refined
m6A MeRIP-seq protocol and analysis pipeline that can be applied to profile low-input
RNA samples from patient
tumors. We optimized the key parameters of
m6A MeRIP-seq, including the starting amount of
RNA,
RNA fragmentation, antibody selection, MeRIP washing/elution conditions, methods for
RNA library construction, and the bioinformatics analysis pipeline. With the optimized immunoprecipitation (IP) conditions and a postamplification rRNA depletion strategy, we were able to profile the
m6A epitranscriptome using 500 ng of total
RNA. We identified approximately 12,000
m6A peaks with a high signal-to-noise (S/N) ratio from 2
lung adenocarcinoma (ADC) patient
tumors. Through integrative analysis of the transcriptome,
m6A epitranscriptome, and
proteome data in the same patient
tumors, we identified dynamics at the
m6A level that account for the discordance between
mRNA and
protein levels in these
tumors. The refined
m6A MeRIP-seq method is suitable for
m6A epitranscriptome profiling in a limited amount of patient
tumors, setting the ground for unraveling the dynamics of the
m6A epitranscriptome and the underlying mechanisms in clinical settings.