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Dataset Persistent ID
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doi:10.34622/datarepositorium/LWNKLQ |
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Publication Date
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2024-07-29 |
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Title
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Endocytosis of the plasma membrane carboxylate transporter Jen1 in Saccharomyces cerevisiae under alkali stress: a mechanism for preventing loss of essential metabolites?
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Author
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Barata-Antunes, Cláudia (Centre of Molecular and Environmental Biology, Department of Biology, University of Minho)
de Hulster, Erik (Department of Biotechnology, Delft University of Technology)
Cardoso, Clara (Department of Biotechnology, Delft University of Technology)
Pronk, Jack (Department of Biotechnology, Delft University of Technology)
Ariño, Joaquín (Institut de Biotecnologia i Biomedicina & Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona)
Mans, Robert (Department of Biotechnology, Delft University of Technology)
Paiva, Sandra (Centre of Molecular and Environmental Biology, Department of Biology, University of Minho)
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Contact
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Use email button above to contact.
Antunes, Cláudia (Centre of Molecular and Environmental Biology, Department of Biology, University of Minho)
Paiva, Sandra (Centre of Molecular and Environmental Biology, Department of Biology, University of Minho)
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Description
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Comparison of mRNA levels by RNA-Seq between Saccharomyces cerevisiae yeast cells grown in steady state, aerobic, continuous cultures growing in synthetic medium with 50 mM ethanol and 30 mM lactate at a dilution rate of 0.07 h-1 and set up at different extracellular pHs (pH 6.0; pH 6.5; pH 7.0; pH 7.1) (2 replicas per different pH - 8 samples in total)
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Subject
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Other
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Keyword
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transporters
carboxylic acids
endocytosis
alkali stress
lactate
pH
chemostat cultivation
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Grant Information
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Fundação para a Ciência e a Tecnologia: PTDC/BIA-MIC/5246/2020
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Depositor
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Barata-Antunes, Cláudia
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Deposit Date
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2024-07-24
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Software
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Illumina plataform
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Characteristic of Sources Noted
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***Growth protocol: Chemostat cultures were grown aerobically at 30 °C, 800 rpm, in synthetic with 33 mM (≈2.9 gL-1) lactate and 50 mM (≈2.3 gL-1) ethanol as sole carbon-limited sources. After the batch phase, the medium pumps were switched on, resulting in the continuous addition of 70 mL/h of SM, supplemented with 33 mM lactate and 50 mM ethanol, to the culture. The working volume (1.0 L) was kept constant using an effluent pump controlled by an electric level sensor, resulting in a dilution rate (D) of 0.07 h−1. The culture pH was maintained at 6.0, 6.5, 7.0 or 7.1 by automated addition of 2 M KOH or 2 M H2SO4. Chemostat cultures were considered to be in steady-state (SS) when, after at least 5 volume changes, the culture dry weight, extracellular metabolite concentrations, the CO2 production and O2 consumption rates did not differ more than 5 % over 2 volume changes. The chemostat runs were limited to less than 15-20 volume changes, after inoculation, to avoid evolutionary adaptation of the cultures*** ***Extracted molecule: Total RNA*** ***Extract protocol: The steady state culture samples (± 240 mg of cells) at the different pHs (6.0, 6.5, 7.0 or 7.1) were collected directly from the bioreactor and instantaneously frozen in liquid nitrogen and stored at – 80 °C for further RNA sample extraction. For RNA extraction, ≈ 800 µL of “crushed ice” bits, from the – 80 °C samples, were transferred to Eppendorf tubes and thawed on ice. Eppendorfs were then centrifuged at 8000 g, 4 min, 0 °C and the pellet was resuspended in 200 µL cold RNase-free water. Subsequently, 400 µL of acid phenol-chloroform was quickly added and the mixture was centrifuged at 8000 g, for 4 min, at room temperature. The aqueous layer was transferred to a new tube and 400 µL of chloroform was added. The mixture was centrifuged again at 8000 g, 4 min, room temperature, and the upper layer was transferred to a new tube. 20 µL of 3 M Na-acetate and 500 µL of 100 % (v/v) ethanol (– 20 °C) were added. After mix by vortexing, the samples were incubated at – 20 °C for at least 15 min. The samples were then centrifuged for 15 min, 4°C, at 8000 g and the pellet was washed with 500 µL 80 % (v/v) ethanol (– 20 °C). The samples were vortexed, centrifuged again and, after removal of all the ethanol, the pellet was resuspended in 20 µL RNase-free water. The samples were kept at room temperature for at least 2 h to dissolve the RNA. The concentration, purity and quality of the total RNA samples were also determined using Tapestation (Agilent Technologies 2200) and NanoDrop 2000 (Thermo scientific)*** ***Library construction: Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.*** ***Data processing: Mapping of paired-end clean reads (2x150 nt) were aligned to the S. cerevisiae CEN.PK113-7D genome (assembly ASM26988v1, obtained from https://fungi.ensembl.org/) with the Hisat2 v2.0.5 software. Percentages of overall alignment were between 92 and 98%. The featureCounts (v1.5.0-p3) program was used to count the number of reads mapped to each gene. Differential expression analysis was conducted using DESeq2 R package (1.20.0) using the Benjamini and Hochberg’s approach to adjust the resulting P-values. Genes with an adjusted P-value <=0.01 found by DESeq2 and a log(2) fold change of +/- 0.6 were assigned as differentially expressed.***
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