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

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

3. 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.

4. 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

5. 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),

6. 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),

7. 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, Sexual Reproduction, Meiosis, and Genetic Recombination. Recovered 18 December 2016 from

8. 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),

9. 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),

10. 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),

11. 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

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

13. 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.