Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.
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.
Earlham Institute, Norwich Research Park, Norwich, UK.
Iain C
IC
Macaulay
Sanger Institute-EBI Single-Cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, UK.|Cancer Genome Project, Wellcome Trust Sanger Institute, Cambridge, UK.
Mabel J
MJ
Teng
Earlham Institute, Norwich Research Park, Norwich, UK.
Wilfried
W
Haerty
Sanger Institute-EBI Single-Cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, UK.|Department of Human Genetics, University of Leuven, KU Leuven, Leuven, Belgium.
Parveen
P
Kumar
Sanger Institute-EBI Single-Cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, UK.|MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, Edinburgh, UK.
Chris P
CP
Ponting
Sanger Institute-EBI Single-Cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, UK.|Department of Human Genetics, University of Leuven, KU Leuven, Leuven, Belgium.
Thierry
T
Voet
D000782:Aneuploidy; D000818:Animals; D002460:Cell Line; D020869:Gene Expression Profiling / Q000379:methods*; D023281:Genomics / Q000379:methods*; D006801:Humans; D051379:Mice; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*
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