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

Abstract

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.

Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

“…Our experience with eCLIP suggests that the input amounts we describe for our protocol could be reduced while still producing quality results, although we have not yet systematically tested the range of adequate RNA input. Consistent with recent reports (Zeng et al 2018), however, we find that the number of unique m 6 A sites identified does increase with higher amounts of input RNA. Therefore, in addition to ensuring accurate quantification of starting material via the methods outlined in our protocol, we also highly encourage the use of multiple replicates when starting RNA material is limited to gain confidence in the identified m 6 A residues.…”

Landscape and regulation of m6A and m6Am methylome across human and mouse tissues

“…To investigate the distribution and dynamics of the m 6 A and m 6 Am among different tissues, transcriptome-wide m 6 A and m 6 Am mapping was performed. Due to the limited amount and partial degraded nature of human tissue RNA samples, we adopted a recently published refined RIP-seq protocol for low-input materials with several modifications (Zeng et al, 2018). It is worth mentioning that instead of random priming, a template-switching reaction at 5’ end of RNA template was used during library construction so that the 5’ end sequence information of RNA was preserved.…”

“…This procedure was performed according to the recently described “Refined RIP-seq” with several modifications(Zeng et al, 2018). 3 μg total RNA was fragmented into ∼130-nucleotide-long fragments by magnesium RNA fragmentation buffer (NEB, E6150S).…”

Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution

“…Typically these approaches require micrograms of polyadenylated starting material (Dominissini et al, 2012;Ke et al, 2015;Linder et al, 2015;Meyer et al, 2012;Schwartz et al, 2013) , which prohibited interrogation of m6A levels in purified populations of cells, clinical settings, or within specific subcellular compartments. While optimized protocols substantially reduced the starting amounts of mRNA (Merkurjev et al, 2018;Zeng et al, 2018) , no protocol exists providing single nucleotide resolution mapping from limited starting mRNA material.…”