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SMARTer single cell total RNA sequencing

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DOI

  • Karen Verboom, Ghent University Hospital, Ghent, Cancer Research Institute Ghent
  • ,
  • Celine Everaert, Ghent University Hospital, Ghent, Cancer Research Institute Ghent
  • ,
  • Nathalie Bolduc, Takara Bio USA, Inc.
  • ,
  • Kenneth J. Livak, Fluidigm Corporation
  • ,
  • Nurten Yigit, Ghent University Hospital, Ghent, Cancer Research Institute Ghent
  • ,
  • Dries Rombaut, Ghent University Hospital, Ghent, Cancer Research Institute Ghent
  • ,
  • Jasper Anckaert, Ghent University Hospital, Ghent, Cancer Research Institute Ghent
  • ,
  • Simon Lee, Takara Bio USA, Inc.
  • ,
  • Morten T. Venø
  • ,
  • Jørgen Kjems
  • Frank Speleman, Ghent University Hospital, Ghent, Cancer Research Institute Ghent
  • ,
  • Pieter Mestdagh, Ghent University Hospital, Ghent, Cancer Research Institute Ghent
  • ,
  • Jo Vandesompele, Ghent University Hospital, Ghent, Cancer Research Institute Ghent

Single cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more limitations, such as focusing only on polyadenylated RNA, sequencing of only the 3' end of the transcript, an exuberant fraction of reads mapping to ribosomal RNA, and the unstranded nature of the sequencing data. Here, we developed a novel single cell strand-specific total RNA library preparation method addressing all the aforementioned shortcomings. Our method was validated on a microfluidics system using three different cancer cell lines undergoing a chemical or genetic perturbation and on two other cancer cell lines sorted in microplates. We demonstrate that our total RNA-seq method detects an equal or higher number of genes compared to classic polyA[+] RNA-seq, including novel and non-polyadenylated genes. The obtained RNA expression patterns also recapitulate the expected biological signal. Inherent to total RNA-seq, our method is also able to detect circular RNAs. Taken together, SMARTer single cell total RNA sequencing is very well suited for any single cell sequencing experiment in which transcript level information is needed beyond polyadenylated genes.

OriginalsprogEngelsk
Artikelnummere93
TidsskriftNucleic Acids Research
Vol/bind47
Nummer16
Antal sider12
ISSN0305-1048
DOI
StatusUdgivet - sep. 2019

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