Biomark hd machine

Differentiations with hiPSC lines were performed in triplicate. For RNA isolations, two pellets were pooled, and isolation was performed as described previously (Bomer et al. 2015 (link)). Total mRNA (150 ng) was processed with a first strand cDNA kit according to the manufacturer’s protocol (Roche Applied Science). cDNA was further diluted five times, and preamplification with TaqMan preamp master mix (Thermo Fisher Scientific Inc.) was performed for a panel of 20 designated genes related to chondrogenesis, hypertrophy, deposition and degradation of cartilage ECM, and neo-cartilage quality (primer sequences in Supplementary Table S1). Gene expression was measured with a Fluidigm Biomark HD machine using a 96.96 IFC chip. Quality control of the data was performed, and non-detected values were imputed according to the minimum detected value. Unsuccessful differentiations, defined by the minimum detected expression of COL2A1 for hPACs and hBMSCs neo-cartilage, were disregarded.

Each DNA sample was tested in one dilution, but two technical replicates were made twice (qPCR mix prepared for four replicates and divided in two aliquots, then DNA was added to both aliquots separately and two inlets were filled from each of the two aliquots). The final volume per inlet was 4 μl (2 μl TaqMan Master Mix, 0.4 μl 20× GE Sample Loading reagent, 0.4 μl of primers and probes mix, and 1.2 μl DNA). Primer and probe concentrations were as in ddPCR reactions.
The reaction mix was loaded on the Fluidigm dPCR 37K IFC chip, inserted into BioMark HD machine (Fluidigm, Markham, Canada), and run with the following program: 2 min at 50 °C, 10 min at 95 °C, and 45 cycles of denaturation and annealing (15 s at 95 °C and 1 min at 60 °C).
Results of estimated targets per panel were used for the calculation of the initial number of target copies present in the stock material. CV was calculated from eight or four replicates, if it was calculated for average of both labs or individual lab, respectively. The experiments were performed in two independent laboratories (NIB and Walloon Agricultural Research Centre).