Single-cell gene expression analysis was performed as described previously [18 (link),57 (link),58 (link)] and as recommended by Fluidigm (http://www.fluidigm.com/single-cell-expression.html ; South San Francisco, CA, USA). Briefly, TS and XEN cells were sorted by fluorescence-activated cell sorting using the MoFlo system (Beckman Coulter, Brea, CA, USA), and individual cells were distributed into wells or 96-well plates containing 5 μl of CellsDirect resuspension buffer (Invitrogen, Carlsbad, CA, USA). The preamplification step consisted of 20 cycles using a mix of universal primer pairs to preamplify each gene simultaneously. Preamplification was followed by exonuclease I treatment (New England Biolabs, Ipswich, MA, USA), and allelic qPCR was performed on a BioMark thermal cycler (Fluidigm). Raw efficiencies of each PCR assay and allelic specificity were measured on control DNA within each experiment. Transcript levels were extrapolated using the raw PCR efficiencies, thus allowing the direct comparison of different genes. Controls for allelic specificities of PCR assays are available upon request. See Additional file 2 A for primer sequences.
Free full text: Click here
