Date of Award

2023

Document Type

Open Access Master's Thesis

Degree Name

Master of Science in Forest Molecular Genetics and Biotechnology (MS)

Administrative Home Department

College of Forest Resources and Environmental Science

Advisor 1

Yinan Yuan

Committee Member 1

Hairong Wei

Committee Member 2

Carsten Kulheim

Abstract

Past transcriptome research on plants focused primarily on protein coding genes, and only recently researchers began looking into the non-protein coding regions that may play significant roles in gene regulation. Antisense RNA transcripts that are found naturally in the cell share complementary sequence with sense transcripts and have been shown to regulate expression of their sense counterparts. Since antisense RNA has been largely under-studied and difficult to sequence because of their low relative abundance, new methods are needed to target antisense RNA for efficient genome-wide profiling. To address this gap in methods to efficiently and cost effectively enrich antisense RNA transcripts for sequencing, we developed methods that allow for the enrichment of antisense RNA through chemically guided annealing of sense/antisense transcript pairs of RNA and cDNA, and the separation of double stranded structures for library formation. These methods utilize guanidinium thiocyanate (GuSCN) to enrich antisense transcripts through the annealing of sense/antisense transcript pairs. Annealed RNA can then be separated through J2 monoclonal anti-dsRNA antibody binding, mRNA purification of enriched transcript pairs, and the custom tailing of sense/antisense transcript pair enriched libraries for Nanopore direct RNA sequencing. We also developed a novel method for the separation of annealed double stranded first strand cDNA using hydroxyapatite powder (HAP) chromatography to form antisense enriched Illumina libraries. The long-term goal of this project is for these methods to be used to form targeted antisense enriched libraries for the genome wide characterization of the antisense RNA response to drought stress in Populus.

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