Refined RIP-seq protocol for epitranscriptome analysis with low input materials

N6-Methyladenosine (m(6)A) accounts for approximately 0.2% to 0.6% of all adenosine in mammalian mRNA, representing the most abundant internal mRNA modifications. m(6)A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) is a powerful technique to map the m(6)A 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 m(6)A 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 m(6)A 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 m(6)A epitranscriptome using 500 ng of total RNA. We identified approximately 12,000 m(6)A peaks with a high signal-to-noise (S/N) ratio from 2 lung adenocarcinoma (ADC) patient tumors. Through integrative analysis of the transcriptome, m(6)A epitranscriptome, and proteome data in the same patient tumors, we identified dynamics at the m(6)A level that account for the discordance between mRNA and protein levels in these tumors. The refined m(6)A MeRIP-seq method is suitable for m(6)A epitranscriptome profiling in a limited amount of patient tumors, setting the ground for unraveling the dynamics of the m(6)A epitranscriptome and the underlying mechanisms in clinical settings.