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Related Concept Videos

RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Full-length RNA-seq from single cells using Smart-seq2.

Simone Picelli1, Omid R Faridani1, Asa K Björklund2

  • 1Ludwig Institute for Cancer Research, Stockholm, Sweden.

Nature Protocols
|January 4, 2014
PubMed
Summary
This summary is machine-generated.

Smart-seq2 enables accurate, full-length RNA sequencing from single cells. This method provides high sensitivity and accuracy for transcriptome analysis, aiding in understanding cell-to-cell variability.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Accurate gene expression quantification in single cells is crucial for understanding cellular heterogeneity.
  • Various high-throughput single-cell RNA sequencing (scRNA-seq) methods exist, differing in coverage, sensitivity, and multiplexing capabilities.

Purpose of the Study:

  • To present a detailed protocol for Smart-seq2, an optimized method for single-cell RNA sequencing.
  • To enable the generation of full-length cDNA and sequencing libraries using standard reagents.

Main Methods:

  • The Smart-seq2 protocol involves cell picking, cDNA synthesis, and library preparation for sequencing.
  • The protocol is designed for high sensitivity, accuracy, and full-length transcript coverage.
  • Utilizes standard laboratory reagents and takes approximately 2 days for library preparation.

Main Results:

  • Smart-seq2 allows for the generation of high-quality sequencing libraries from individual cells.
  • The method achieves improved sensitivity and accuracy compared to previous iterations.
  • Provides full-length coverage across transcripts, offering comprehensive transcriptome analysis.

Conclusions:

  • Smart-seq2 is a robust and efficient protocol for single-cell RNA sequencing.
  • The method facilitates detailed analysis of cell-to-cell variability in gene expression.
  • Current limitations include lack of strand specificity and inability to detect non-polyadenylated RNA.