Biomark analysis system

Single-cell gene expression analysis was performed using BioMark 96.96 Dynamic Array platform (Fluidigm, San Francisco, CA) and TaqMan Gene Expression Assays (Applied Biosystems, Carlsbad, CA). Single cells were sorted into 5μl of CellsDirect reaction mix and immediately stored in -80C. Control wells containing no cells were included. On thawing, a mix containing 2.5μL gene specific 0.2x TaqMan Gene Expression Assays (Applied Biosystems), 1.2 μL CellsDirect RT/Taq mix, and 0.3 μL TE buffer were added to each well. RT–PCR pre-amplification cycling conditions were: 50°C, 15min; 95°C, 2min; 22x(95°C, 15s; 60°C, 4min). Samples were diluted 1:5 in TE buffer and 6% were mixed with TaqMan Universal PCR Master Mix (Applied Biosystems). The sample mix and TaqMan assays were loaded separately into the wells of 96.96 Gene expression Dynamic Arrays (Fluidigm) in presence of appropriate loading reagents. The arrays were read in a Biomark analysis system (Fluidigm). ΔCt values were calculated in reference to the average of Atp5a1, Hprt1 and Ubc.
The expression of each gene was fit by a bivariate normal distribution (R mixtools package [22 ]) and a cut-off set at the average position between the lower and upper gaussian midpoints. Genes above this were considered expressed if above this cut-off. qPCR levels and metadata are provided in S1 File.

Single-cell gene expression analysis was performed using the BioMark 96.96 Dynamic Array platform (Fluidigm, San Francisco, CA, USA) and TaqMan gene expression assays (Applied Biosystems, Carlsbad, CA, USA). We sorted 48 single cells for each of the four populations into 5 μl of CellsDirect reaction mix and these were immediately stored at −80 °C. Control wells containing no cells were included. Upon thawing, a mix containing 2.5 μL gene-specific 0.2× TaqMan gene expression assays (Applied Biosystems), 1.2 μL CellsDirect RT/Taq mix and 0.3 μL TE buffer were added to each well. RT-PCR pre-amplification cycling conditions were: 50 °C, 15 min; 95 °C, 2 min; 22 × (95 °C, 15 s; 60 °C, 4 min). Samples were diluted 1:5 in TE buffer and 6 % were mixed with TaqMan Universal PCR Master Mix (Applied Biosystems). The sample mix and TaqMan assays were loaded separately into the wells of 96.96 Gene Expression Dynamic Arrays (Fluidigm) in the presence of appropriate loading reagents. The arrays were read in a Biomark analysis system (Fluidigm). ΔCt values were calculated in reference to the average of Atp5a1, Hprt1 and Ubc. A list of the genes analysed can be found in Additional file 2: Table S6.