Project description:Supplement S1: Figure S1. (a) Effect of culture age of Euglena gracilis on gene expression, determined with gene arrays analysis. Total number of differentially expressed genes classified into groups of fold-change-ranges in Euglena gracilis cells of different culture age (5 d, 9 d, 11 d) compared to culture age of six days. Underlying data are presented under the figure or in “FC in classes”, respectively. The total number of investigated genes on microarray was n = 20296. Data were retrieved from four independent cell cultures. (b) Effect of culture age of Euglena gracilis on gene expression. Fraction of differentially expressed genes in percent of all investigated transcripts [n = 20,396] classified into groups of fold-change-ranges in Euglena gracilis cells of different culture age (5 d, 9 d, 11 d) compared to culture age of six days. Underlying data are presented under the figure or in “FC in classes”, respectively. For more details, please see the legend Figure S1 a. (c) Effect of culture age of Euglena gracilis on gene expression. Fraction differentially expressed genes (DEGs) above (“upregulation”), below (“downregulation”) or above AND below (“all”), respectively a certain threshold (th). In contrast to (b), where the fold changes are provided in classes, all DEGs above, below, or above AND below as certain threshold are summarized. In addition, Supplement S1 contains all raw data about all DEGs detected and a rankling of the genes regarding their fold changes (FCs). Supplement S2: Determination of differential gene expression with respect to culture age in Euglena gracilis, obtained with micro-arrays. The table “Overlapping DEGs” contains all transcripts, which were found to be differentially expressed in Euglena gracilis at all three investigated days of culture age (5, 9, or 11 days, respectively), against the 6-day samples. In the table “Overlapping DEGs ranked”, the data are ranked according to their fold change. Supplement S3: Transcription of Euglena gracilis cells of different culture age (5, 9, and 11 d, respectively) was compared with the transcription of cells after 6 days of culturing. All data were obtained in quadruplicate. This supplementary data show the possible functions of the proteins encoded by the DEGs (differentially expressed genes), which were differentially expressed for the corresponding days. Table “Diff transcripts funct.” gives a general overview about gene expression changes for all days. The other tables list the possible function of the DEGs for each particular day. The lists only contain genes where a function could be assigned. Complete lists of DEGs also including transcripts with unknown function are provided in Supplement S1. Supplement S4: Determination of gene expression changes with respect to culture age in Euglena gracilis samples of cells were analyzed after 5 d, 9 d, and 11 days of growth in batch cultures, and their gene expression was compared with the gene expression of cells with a culture age of 5 d. For each day, the number of differentially expressed genes (DEGs) each showing the same GO terms were summed up. In order to correlate these changes with the number of all genes with the same GO term in Euglena gracilis, data from sequencing projects were employed [1,2]. Genes with the same GO terms were counted and correlated with the number of observed DEGs in Euglena gracilis. In the first table, GO terms of DEGs were assigned to corresponding days of culturing and correlated with the data of [1, 2] (total numbers and ratios). Diagram “Ratio of DEGs compared to Cordoba et al. [%]” visualizes the ratio of DEGs with total data of [7]. The second diagram shows the number of GO terms of DEGs, which were found at day 9 AND day 11. The change between the two days is provided. Data are visualized in the diagram “Fraction of DEGs of a certain GO over time”. Ref. [2]: Additional file 2: Table S1 “proteome”. Ref. [1]: Supplementary Data, Table S2. Supplement S5: Amino acid sequences of proteins, which were found to become differentially expressed with respect to culture age of Euglena gracilis cultures. [1] Cordoba, J.; Perez, E.; Van Vlierberghe, M.; Bertrand, A.R.; Lupo, V.; Cardol, P.; Baurain, D. De novo transcriptome meta-assembly of the mixotrophic freshwater microalga Euglena gracilis. Genes 2021, 12, 842. https://doi.org/10.3390/genes12060842. [2] Ebenezer, T.E.; Zoltner, M.; Burrell, A.; Nenarokova, A.; Novák Vanclová, A.M.G.; Prasad, B.; Soukal, P.; Santana-Molina, C.; O’Neill, E.; Nankissoor, N.N.; et al. Transcriptome, proteome and draft genome of Euglena gracilis. BMC Biol. 2019, 17, 11. https://doi.org/10.1186/s12915-019-0626-8.

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