Pilar Bustamante Madrid Single-cell RNA-seq Pilar Bustamante Madrid

Index Introduction Methodology Isolation of single cell Single-cell RNA-seq Applications of single-cell RNA-seq Future challenges

INTRODUCTION Global studies of a single cell has been favour by the high sensitivity and automatization of instruments. Analysis of single cell transcriptomes by RNA-seq to profile cell-to-cell viability.

INTRODUCTION RNA-seq RNA plays diverse roles so it can reveal the state of the cell on a particular moment. Analysed of RNAs and different genes expression level on different exposures to external factors or during the development 1. Isolation of individual cells of interest 2. Converting the extracted RNA into cDNA and sequence it

METHODOLOGY Isolation of a single-cell from a potential heterogeneous population. (A-C isolation from a cell suspension and D from tissue samples) Saliba, A.E., Westermann, A.J., Gorski, S.A., and Vogel, J. (2014). Single-cell RNA-seq: advanced and future challenges. Nucleic acids research, 42(14), 8845-8860.

METHODOLOGY Isolation of a single-cell from a potential heterogeneous population. (A-C isolation from a cell suspension and D from tissue samples) Saliba, A.E., Westermann, A.J., Gorski, S.A., and Vogel, J. (2014). Single-cell RNA-seq: advanced and future challenges. Nucleic acids research, 42(14), 8845-8860.

Reverse transcription METHODOLOGY 2. Single-cell RNA-seq Reverse transcription The strategies to synthetized the second strand of cDNA are one of the followings ones: Addison Wesley Longman,. 1999. Sexual Reproduction, Meiosis, and Genetic Recombination. Recovered 18 December 2016 from http://www.mun.ca/biology/desmid/brian/BIOL4900/CB18_31.html#

METHODOLOGY 2. Single-cell RNA-seq Saliba, A.E., Westermann, A.J., Gorski, S.A., and Vogel, J. (2014). Single-cell RNA-seq: advanced and future challenges. Nucleic acids research, 42(14), 8845-8860.

METHODOLOGY 2. Single-cell RNA-seq Saliba, A.E., Westermann, A.J., Gorski, S.A., and Vogel, J. (2014). Single-cell RNA-seq: advanced and future challenges. Nucleic acids research, 42(14), 8845-8860.

METHODOLOGY 2. Single-cell RNA-seq  Template-switching and In vitro transcription mechanism maintain the full length and strand specific information Saliba, A.E., Westermann, A.J., Gorski, S.A., and Vogel, J. (2014). Single-cell RNA-seq: advanced and future challenges. Nucleic acids research, 42(14), 8845-8860.

APPLICATIONS APPLICATIONS How the data is interpret and make sense cell-to-cell variability? How can variability between cells with the same genetic context be explained? APPLICATIONS

Non-represented by RNA-seq FUTURES CHALLENGES Non-represented by RNA-seq RNA prokaryote sRNA microRNA Solution: Not so random primers Nanopore

CONCLUSION RNA-seq is used to know the qualitative and quantitative gene expression. A major future challenge will be to go beyond poly(A) transcriptome of eukaryote and bring single cell to the level that all types of cellular transcript are analysed in parallel. The long term goal must be to directly sequence full length RNA molecules.