HANSOLL NA
Research on Detection of Previously Unknown RNA Modification
During my master's degree, I also worked on another project focused on detecting previously unknown RNA modifications (hereinafter unkMOD) using Oxford Nanopore direct RNA sequencing.
In recent years, various post-transcriptional RNA modifications, such as m6A methylation, have been studied using experimental approaches relying on high-throughput sequencing. Emerging evidence links RNA modifications to cellular processes, including cell proliferation, and physiology of disease due to their widespread influence. Given this widespread influence, numerous studies have explored those by harnessing Oxford Nanopore direct RNA sequencing, which enables direct examination of modifications within individual transcripts. I also employed this cutting-edge technique in this research.
In collaboration with the other lab, preliminary results revealed that the presence of unkMOD on RNA detected using a specific antibody. Once the existence of these modifications on RNA was confirmed through immunoblot assay, I embarked on using direct RNA sequencing to address key questions:
1. What types of RNAs undergo this modification?
2. Is there a specific motif or residue to which this modification is attached to RNA molecules?
3. What are the functional roles of this modification on RNA?
These inquiries guided the next phase of the research.
My strategy for addressing these questions compares control and knockdown samples. In the knockdown sample, a specific gene X associated with this modification was silenced using siRNA. This experimental design aims to identify modified RNAs and the modified residues by detecting differences in signals between the control and knocked-down samples.
Following the strategy I devised, I constructed the library from scratch. Firstly, I used siRNA to silence gene X in a human cell line. Secondly, I isolated total RNA from both control and knockdown samples, and conducted poly A selection. Lastly, I meticulously prepared the direct RNA sequencing library following ONT's protocol. Throughout these procedures, I encountered and addressed various troubleshooting challenges, optimizing the library preparation. With the final protocol, I successfully performed Nanopore direct RNA sequencing. I achieved a high read throughput of over 1M reads with R9.4.1 and MinION, despite the slower sequencing speed of RNA compared to DNA.