scRNA-Seq
Single-Cell mRNA Sequencing
scRNA-Seq provides deeper insight to the multi-tiered complexity of different cells within the same tissue type (Tang et al., 2009). scRNA-Seq has now been adapted widely into other methods in the single-cell RNA sequencing field.In this method, single cells are isolated manually under a microscope and lysed. Next, mRNAs are purified and primed with a poly(T) primer for reverse transcription. Unreactive primers are removed by exonuclease I digestion. Poly(A) tails are added to the first strand cDNA at the 3′ end and annealed to poly(T) primers for second-strand cDNA generation. Finally, the cDNAs are PCR-amplified, sheared, and prepared into sequencing libraries.
Advantages:
- Single-cell_resolution transcriptomic analysis
- Able to detect unknown splice junctions
Disadvantages:
- Very low throughput
- No molecular barcodes used
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Moignard V. and Gottgens B. Dissecting stem cell differentiation using single cell expression profiling. Curr Opin Cell Biol. 2016;43:78-86
Woodhouse S., Moignard V., Gottgens B. and Fisher J. Processing, visualising and reconstructing network models from single-cell data. Immunol Cell Biol. 2016;94:256-265
Wagner A., Regev A. and Yosef N. Revealing the vectors of cellular identity with single-cell genomics. Nat Biotechnol. 2016;34:1145-1160
Stegle O., Teichmann S. A. and Marioni J. C. Computational and analytical challenges in single-cell transcriptomics. Nat Rev Genet. 2015;16:133-145
Kolodziejczyk A. A., Kim J. K., Svensson V., Marioni J. C. and Teichmann S. A. The Technology and Biology of Single-Cell RNA Sequencing. Mol Cell. 2015;58:610-620
References:
Lee J. H., Daugharthy E. R., Scheiman J., et al. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat Protoc. 2015;10:442-458
Padovan-Merhar O., Nair G. P., Biaesch A. G., et al. Single Mammalian Cells Compensate for Differences in Cellular Volume and DNA Copy Number through Independent Global Transcriptional Mechanisms. Mol Cell. 2015;58:339-352
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History: scRNA-Seq
Revision by sbrumpton on 2017-06-21 07:50:22 - Show/Hide
Single-Cell mRNA Sequencing
scRNA-Seq provides deeper insight to the multi-tiered complexity of different cells within the same tissue type (Tang et al., 2009). scRNA-Seq has now been adapted widely into other methods in the single-cell RNA sequencing field.In this method, single cells are isolated manually under a microscope and lysed. Next, mRNAs are purified and primed with a poly(T) primer for reverse transcription. Unreactive primers are removed by exonuclease I digestion. Poly(A) tails are added to the first strand cDNA at the 3' end and annealed to poly(T) primers for second-strand cDNA generation. Finally, the cDNAs are PCR-amplified, sheared, and prepared into sequencing libraries.
Advantages:- Single-cell_resolution transcriptomic analysis
- Able to detect unknown splice junctions
Disadvantages:- Very low throughput
- No molecular barcodes used
Reagents:Illumina Library prep and Array Kit SelectorReviews:Moignard V. and Gottgens B. Dissecting stem cell differentiation using single cell expression profiling. Curr Opin Cell Biol. 2016;43:78-86Woodhouse S., Moignard V., Gottgens B. and Fisher J. Processing, visualising and reconstructing network models from single-cell data. Immunol Cell Biol. 2016;94:256-265Wagner A., Regev A. and Yosef N. Revealing the vectors of cellular identity with single-cell genomics. Nat Biotechnol. 2016;34:1145-1160Stegle O., Teichmann S. A. and Marioni J. C. Computational and analytical challenges in single-cell transcriptomics. Nat Rev Genet. 2015;16:133-145Kolodziejczyk A. A., Kim J. K., Svensson V., Marioni J. C. and Teichmann S. A. The Technology and Biology of Single-Cell RNA Sequencing. Mol Cell. 2015;58:610-620References:Lee J. H., Daugharthy E. R., Scheiman J., et al. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat Protoc. 2015;10:442-458Padovan-Merhar O., Nair G. P., Biaesch A. G., et al. Single Mammalian Cells Compensate for Differences in Cellular Volume and DNA Copy Number through Independent Global Transcriptional Mechanisms. Mol Cell. 2015;58:339-352