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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. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing
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An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

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High-throughput full-length single-cell RNA-seq automation.

Lira Mamanova1, Zhichao Miao2,3, Ayesha Jinat2

  • 1Wellcome Sanger Institute, Cambridge, UK. lm4@sanger.ac.uk.

Nature Protocols
|May 15, 2021
PubMed
Summary
This summary is machine-generated.

We automated full-length single-cell RNA sequencing protocols, enhancing scalability and reducing costs. This makes high-resolution transcriptomic analysis more accessible for rare cell studies.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Full-length single-cell RNA sequencing (scRNA-seq) offers high transcriptomic complexity, sensitivity, and specificity.
  • Advantages include isoform analysis, SNP detection, and VDJ region assembly for T- and B-cell receptors.
  • Current plate-based full-length protocols are costly and less scalable, limiting their widespread adoption compared to droplet or nanowell methods.

Purpose of the Study:

  • To develop and optimize automated protocols for full-length single-cell RNA sequencing.
  • To improve the scalability and reduce the cost of full-length scRNA-seq.
  • To provide accessible workflows for researchers, including those with limited automation experience.

Main Methods:

  • Description of an in-house automated Smart-seq2 protocol and a commercial kit-based workflow.
  • Benchmarking of lysis buffers and reverse transcription enzymes to optimize the in-house protocol.
  • Evaluation of protocols based on ease of use, equipment needs, processing time, cost, and sequencing quality.

Main Results:

  • Developed automated workflows for full-length scRNA-seq, completing in 3-5 days.
  • Optimized the in-house protocol, significantly reducing experimental costs.
  • Demonstrated successful application of these automated pipelines in Human Cell Atlas initiative projects.

Conclusions:

  • Automated full-length scRNA-seq protocols enhance scalability and reduce costs, making them more accessible.
  • These optimized protocols are suitable for profiling rare cell types and can be adopted by researchers with basic lab skills.
  • The developed methods facilitate high-resolution transcriptomic studies and support large-scale initiatives like the Human Cell Atlas.