Supplemental Fig. 1 Diagram depicting the setup of the SRIS experiments showing the spore and the micro-stepper motor. The single probe can be operated at only on position and the imaging is done using either the video-zoom microscope, or the inverted microscope, but not concurrently.
Supplemental Fig. 2 Four of the wells on one CEL-C chip. In this picture each well is loaded with one C. richardii spore. One Ag/AgCl calcium specific electrode leads to each of the four positions (top, bottom and two sides) around each well. Each CEL-C chip has 16 such wells. Scale bar is 300 microns.
Supplemental Fig. 3 Early RNA expression of CrACA1 in germinating spores.
RNA was isolated from spores collected every three hours after light exposure through 24 h. Real time RT-PCR was performed as described below using the LUX™ primer system with APT1 as a reference gene. Changes in concentration were normalized to hour 0 using the standard curve method. Error bars represent 95% confidence intervals calculated as described. Non-overlapping intervals indicate statistically significant expression differences. Reactions were run on a 7900HT Sequence Detector machine (Applied Biosystems; Foster City, CA) as an absolute quantification run. PCR reactions were performed in 96-well polypropylene microplates using Platinum® Quantitative PCR SuperMix-UDG polymerase master mix (Invitrogen) at one-half final volume (25 µL) according to the Invitrogen cycling programs and protocols. A final concentration of 50 nM of appropriate gene-specific primers and the equivalent of 100 ng of reverse-transcribed RNA were used per reaction. Reaction conditions used were: 50°C for 2 min.;95°C for 2 min.;40 or 45 cycles of 95°C for 15 s, 55°C for 30 s, 72°C for 30 s, followed by the software’s default melting curve analysis. Using previously established guidelines (Cantero et al., 2006), a minimum of six useable data points were taken from at least two technical replicates of three biological replicates. The actual n value for each time point was eight or nine. Standard curves were generated from five-fold serial dilutions of cDNA pooled from all time points.
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