Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing
| Autor: | Jennifer N Dumdie, Srimeenakshi Srinivasan, Heidi Cook-Andersen, Robert Morey, Soheila Vaezeslami, Sergio Mora-Castilla, Cuong To, Louise C. Laurent, Joby Jenkins |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
DNA Complementary Library preparation Cell Sequencing data Biomedical Engineering Computational biology Biology DNA sequencing Analytical Chemistry miniaturization 03 medical and health sciences 0302 clinical medicine Single-cell analysis Complementary Complementary DNA Genetics medicine Humans Embryonic Stem Cells Original Research Computational Biology High-Throughput Nucleotide Sequencing library scale-down DNA Stem Cell Research Molecular biology Computer Science Applications Transcriptome Sequencing single cell Medical Laboratory Technology 030104 developmental biology medicine.anatomical_structure Generic health relevance Single-Cell Analysis Scale down 030217 neurology & neurosurgery Biotechnology |
| Zdroj: | Mora-Castilla, Sergio; To, Cuong; Vaezeslami, Soheila; Morey, Robert; Srinivasan, Srimeenakshi; Dumdie, Jennifer N; et al.(2016). Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing.. Journal of laboratory automation, 21(4), 557-567. doi: 10.1177/2211068216630741. UC San Diego: Retrieved from: http://www.escholarship.org/uc/item/9n172726 Journal of laboratory automation, vol 21, iss 4 Slas Technology |
| DOI: | 10.1177/2211068216630741. |
| Popis: | As the cost of next-generation sequencing has decreased, library preparation costs have become a more significant proportion of the total cost, especially for high-throughput applications such as single-cell RNA profiling. Here, we have applied novel technologies to scale down reaction volumes for library preparation. Our system consisted of in vitro differentiated human embryonic stem cells representing two stages of pancreatic differentiation, for which we prepared multiple biological and technical replicates. We used the Fluidigm (San Francisco, CA) C1 single-cell Autoprep System for single-cell complementary DNA (cDNA) generation and an enzyme-based tagmentation system (Nextera XT; Illumina, San Diego, CA) with a nanoliter liquid handler (mosquito HTS; TTP Labtech, Royston, UK) for library preparation, reducing the reaction volume down to 2 µL and using as little as 20 pg of input cDNA. The resulting sequencing data were bioinformatically analyzed and correlated among the different library reaction volumes. Our results showed that decreasing the reaction volume did not interfere with the quality or the reproducibility of the sequencing data, and the transcriptional data from the scaled-down libraries allowed us to distinguish between single cells. Thus, we have developed a process to enable efficient and cost-effective high-throughput single-cell transcriptome sequencing. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|