Total mRNA expression in individual cells A developed method for isolation and total transcriptome analysis of single cells

Abstract

Methods to analyze individual cells are highly desired to discover important varia-tions and mechanisms that can not be found when studying a bulk population of cells. Insight in single cell variations can help us find answers to important ques-tions about for example tumor development and drug resistance among tumor cells. Transcriptomics is one branch within the field of single cell analysis with the purpose of analyzing the gene expression in one cell. This thesis will present a new approach to perform total transcriptome measure-ments of single cells. The approach includes isolation of individual cells using the automatic cell picking system CellCelector followed by full length reverese transcrip-tion and global preamplification of the total mRNA exposed from direct lysis. The method is based on the Smart-seq2 protocol [1] used to generate full length cDNA and sequencing libraries from single cells. The developed method has been applied on two scenarios to try to answer if the total transcriptome expression is a˙ected in: • Tumor cells expressing the fusion oncogene FUS-DDIT3. • Cells exposed to serum starvation The proposed method has in this project been shown promising as a tool to measure the total transcriptome in single cells. Singe cells variations have been observed and indications of a lower total transcriptome expression in serum starved have been discovered. Though, it seems like the FUS-DDIT3 fusion oncogene does not e˙ect the total transcriptome in single cells.