Análisis de Confiabilidad General del Instrumento

In attempt to replicate Houseley et al. results, we retrieved experimental conditions analogous to theirs: wild-type and GAL10-lncRNAΔ cells were cultured overnight in YEP media with 2% raffinose, diluted in the morning in fresh pre-warmed media to an OD600 of 0.05, and loaded into the

microfluidic device at the beginning of the log-mid phase growth (OD600 ~ 0.2). The adopted device

was a modified version of ALCATRAS - A Long-term Culturing And TRApping System [160] – which, consisting of three chambers, allows the simultaneous monitoring of up to three strains subjected to the same media conditions. Once loaded, cells experienced YEP with 2% raffinose, 0.01% galactose and 0.02% glucose over the 20 hours of imaging. While the loading protocol, set by the device structure, impeded the precise identification of the instant at which cells first sensed the partially inducing media, efforts were taken to constrain it to the 20 minutes preceding the acquisition start. The results, shown in Figure 4.3, are in qualitative agreement with those published by Houseley et al., reproducing a higher fluorescence for the GAL10-lncRNAΔ strain over the first 5 hours of induction. Considering the constant supply of fresh media during the experiment, the comparable fluorescent levels observed in the following 4 hours could not be attributed to the authors paved hypothesis of a decrease in batch cultures cellular growth rate at high OD600, where the nutrients

content might become a limiting factor. Finally, the data revealed the appearance of a tardive difference in GAL1 expression among the tested strains, which could reflect the GAL pathway activation. In that case, the unusually long lag time could be ascribed to both the inability to detect faint initial differences in fluorescent signal, due to the high autofluorescence of YEP media, and the low galactose concentration, whose activating effect is moreover dampen by the presence of glucose traces.

The late fluorescent boost was never observed in subsequent experiments, although it might reflect a cellular behaviour liable to investigation with acquisition of extended duration. Such experiments present technical challenges in ensuring cell fitness.

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Although the results of the above experiment were in line with our expectations, subsequent repeats revealed poor reproducibility, providing data set in which GAL1 expression levels of the analysed strains were hardly distinguishable. We reasoned that the originally revealed qualitative agreement indicated our proximity to a nutrients environment in which strain-dependent differences in GAL1 expression could be detected.

We therefore employed flow cytometry analysis to screen transformants behaviour when exposed to slightly different mixes of sugars in Synthetic Complete (SC) media, usually selected for the preparation of liquid yeast cultures committed to fluorescent measurements because of its reduced autofluorescence compared with other richer media, such as XY or YPD.

We grew overnight wild-type cells and GAL10-lncRNAΔ mutants in SC media with 2% raffinose (30°C, 200rpm). In the morning, cell cultures were diluted to an OD600 of 0.05 in fresh pre-warmed raffinose

media and further incubated until they reached an OD600 of 0.2, at the beginning of log phase growth.

Samples harvested from these were hence centrifuged and the pellet resuspended in an equal volume of SC containing 2% raffinose, 0.02% glucose and galactose concentrations equal to 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%. Two hours later, samples were assayed at the flow cytometer to quickly measure the fluorescent distribution (~2x104 _{fluorescent events) of the two transformants.}

Each sample was acquired twice, and, given that the cells were not fixed, the order adopted for Figure 4.3: Results of wild-type and GAL10-lncRNA cells, induced in accordance with Houseley et al. experimental conditions (SC with 2% raffinose, 0.01% galactose and 0.02 %) using the microfluidic device. The results refer to the 433 wild-type and 488 GAL10-lncRNA cells that remained present for more than 220 time points. In panel A the mean fluorescence over the whole acquisition is shown for wild-type (blue) and GAL10-lncRNA (red) population, with shaded area representing the standard error on the mean. The sharp depression in wild-type mean fluorescence 2 hours after the beginning of the acquisition is due to a loss of focus. As detailed in the main text, the comparison of populations behaviour qualitatively indicate a faster and stronger activation for GAL10-lncRNA strain. Ten hours after the beginning of induction the high fluorescence boost, more prominent for wild-type cells, is observed. Panel B shows experimental data (mean ± standard error) extracted, for both strains, at the same time-points at which RNA samples were harvested in Houseley et al. paper. In this case the mean fluorescence value at the first time point was subtracted from all data. The comparison of panels B and C, where Houseley et al. [3] results are reproduced with permission, denotes a general agreement in the two set of experiments.

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samples test was reversed among the two runs to compensate for distortions in results due to temporal bias during the transient activation.

Among the assayed conditions, 0.04% and 0.06% galactose showed a robust and statistically significant difference in GAL1 expression between wild-type and GAL10-lncRNAΔ strains. The first one (Figure 4.4), being closer to the 0.01% galactose originally adopted by Houseley et al. [3], was selected for the subsequent experiments.

Figure 4.4: Comparison of fluorescent histograms plot of wild-type (blue line) and GAL10- lncRNAred linecells exposed to SC containing 2% raffinose, 0.02% glucose and 0.04% galactose. On the y axes the number of fluorescent events is normalized to the whole set of acquired ones. The histograms were found to be statistically different (p-value< 10-5_{) by the two samples KS test (α=}

5%)Among the tested inducing media providing robust and discernible expression, this one was selected as having the galactose concentration closest to the one adopted in Houseley et al.

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4.3 Induction of wild-type and GAL10-lncRNAΔ strains with