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Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq

Abstract

Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in 3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

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Figure 1: Stepwise overview of the G&T-seq method.
Figure 2: Representative Agilent Bioanalyzer electropherogram plots from various stages of single-cell RNA and DNA amplification and library preparation.
Figure 3: Single-cell DNA-copy-number profiles.
Figure 4: Transcript detection in G&T-seq data from HCC38 and HCC38-BL cell lines.

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Acknowledgements

This work was supported by the Wellcome Trust (grants to T.V. & C.P.P.) and funding from the Research Foundation Flanders (FWO) and the KU Leuven to T.V. (FWO–G.0687.12; KU Leuven SymBioSys, PFV/10/016). W.H. and C.P.P. were also funded by the Medical Research Council. M.J.T. is supported by a Wellcome Trust Sanger Institute Clinical PhD fellowship (UK).

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Authors

Contributions

I.C.M., C.P.P. and T.V. devised and developed the method and wrote the manuscript. M.J.T. assisted with method development. W.H. and P.K. performed bioinformatics analysis of data.

Corresponding authors

Correspondence to Iain C Macaulay or Chris P Ponting or Thierry Voet.

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Competing interests

T.V. is co-inventor on patent applications: WO/2011/157846, ‘Methods for haplotyping single cells’; WO/2014/053664, ‘High-throughput genotyping by sequencing’; and WO/2015/028576, ‘Haplotyping and copy number typing using polymorphic variant allelic frequencies’.

Integrated supplementary information

Supplementary Figure 1 Example robot deck layout for separation of DNA and RNA (steps 17-28)

Panel A shows the layout of the deck on the Beckman FxP robot for this process. Panel B shows a pictorial view of the deck layout with plates and tip types indicated.

Supplementary Figure 2 Example robot deck layout for separation of DNA and RNA (steps 47-55)

Panel A shows the layout of the deck on the Beckman FxP robot for this process. Panel B shows a pictorial view of the deck layout with plates and tip types indicated.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 314 kb)

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Macaulay, I., Teng, M., Haerty, W. et al. Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq. Nat Protoc 11, 2081–2103 (2016). https://doi.org/10.1038/nprot.2016.138

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