Abstract 3728: A novel, simplified, streamlined workflow for high-throughput whole transcriptome RNA-seq library preparation for a variety of sample types

Abstract

Abstract RNA sequencing (RNA-seq) has become an invaluable tool in the study of biology, but often requires multiple days to process samples from total RNA to final libraries that can be loaded on to a sequencer. Researchers use RNA-seq to gain insight into whole tumor gene expression, and in combination with other omics data make treatment decisions. In cancer research, time taken to yield sequencing results can be critical for clinical samples. Within oncology research, libraries are often prepared from RNA sourced from formalin-fixed, paraffin-embedded (FFPE) tissues which can be particularly challenging and is not compatible with all library preparation methods. Here, we present a new protocol that allows users to easily generate robust RNA-seq libraries from a variety of sample types in a single day from 25-250 ng of total RNA. Compatible with poly(A) enrichment or ribosomal RNA depletion, this new workflow has reduced preparation time by about 40% compared to current library prep kits without sacrificing quality of libraries or any sequencing metrics, including 5’-3’ coverage, GC bias. Our new method provides equivalent transcript expression in FFPE samples when compared with existing protocols. In addition to master mixed components and reduced incubation times, the reduction in bead cleanup steps significantly reduces hands-on time for library preparation, simplifying and streamlining the process. This new library preparation method is also ideal for processing samples of various inputs with a single condition at every step throughout the protocol, without modifying cycling conditions or dilutions based on input amount. This workflow provides a great advantage in efficiently generating RNA-seq libraries while simultaneously maintaining high quality sequencing results and compatibility with both high- and low-quality RNA such as FFPE. Citation Format: Chelsea Pinegar, Dora Posfai, Gautam Naishadham, Bradley W. Langhorst, Keerthana Krishnan. A novel, simplified, streamlined workflow for high-throughput whole transcriptome RNA-seq library preparation for a variety of sample types. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3728.