Combined sequencing of mRNA and DNA from human embryonic stem cells.
Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471.
Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Ihnestr. 63-73, 14195 Berlin, Germany.
Florian
F
Mertes
Next Generation Sequencing Group, Max Planck Institute for Molecular Genetics, Ihnestr. 63-73, 14195 Berlin, Germany.
Heiner
H
Kuhl
Institute for Stem Cell Research and Regenerative Medicine, Heinrich Heine University, Moorenstr. 5, 40225 Düsseldorf, Germany.
Wasco
W
Wruck
Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Ihnestr. 63-73, 14195 Berlin, Germany.
Hans
H
Lehrach
Institute for Stem Cell Research and Regenerative Medicine, Heinrich Heine University, Moorenstr. 5, 40225 Düsseldorf, Germany.
James
J
Adjaye
Embryonic stem cells; Next generation sequencing; RNA and whole-genome sequencing; Single cell; Ultra-low input sequencing