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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.

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