Advances on the detection of N6-methyladenosine (m6A) modifications in glioma stem cells / Rugilė Dragūnaitė

Experimental medicine

Bibliogr.: p. 32

Introduction Rapid development of RNA genome sequencing methods over the last decade has revolutionized in cancer biology. Next generation sequencing (NGS) has become the standart in research and public health [1] and is a key technique used for detection of post-trancriptional RNA modifications (i.e., N6 -adenosine methylation (m6A)). RNA-seq technology was utilized to uncover m6A modification in glioma stem cells (GSCs) [2] and the evidence is emerging that m6A commands self-renewal, tumorigenesis and traditional therapy resistance of GSCs [3].Aim The aim of this study was to analyze the advantages and disadvantages in RNA m6Adetection using Illumina and Nanopore. Methods For the Illumina RNA-seq fragmented poly A enriched RNA was used RIP-se q protocol (Novogene, Europe) where RNA was synthesized to double-strand cDNA and PCR amplified. For data analysis, the pipeline “nfcore/chip-seq” (ver.: 1.2.2) was applied to detect m6Amodifications. For the Nanopore RNA sequencing was used native poly A RNA and library was prepared using ”Direct RNA sequencing Kit (SQK-RNA002, Oxford Nanopore Technologies)according to the manufacturer’s instructions. For the RNA modifications detection was used “nf-core/nano seq” (ver.:2.0.1) pipeline. Results In this study, we analyzed that both Illumina and Nanopore has its own techniques to detect the sequence in molecules. One of the main differences between Illumina and Nanopore is that Illumina RNA library is formed by poly A capture and reverse trancription of cDNA while Nanopore requires first-strand DNA synthesis from native poly A RNA. Next, fort he Illumina about 500 µg total RNA was used whereas about 40µg total RNA was used for the Nanopore. Morever, Nanopore presents qualitative m6A data (indicating probability of the site to be modified) while Illumina shows [...].