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

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 conditions and a post-amplification rRNA depletion strategy, we were able to profile the m6A epitranscriptome using 2 µg of total RNA. We identified ~12,000 m6A peaks with a high signal/noise ratio from two 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 accounts for the discordance between mRNA and protein levels in these tumors. Refining MeRIP-seq by comparing 3 antibodies and 5 different amounts of staring materials in A549 cells line, in which 0.5ug library is with 2 replicates. Refined MeRIP-seq were applied to 2 lung Adenocarcinoma patient tumors