HxOH treatment of Synechocystis culture
Differential gene expression analysis (DESeq2) of Synechocystis culture treated with 20 mM of hexan-1-ol for 30 min in 3 independent replications for each, "-" and "+" samples.
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In our previous study, we showed that, among other primary aliphatic alcohols, hexan-1-ol is characterized by the strongest effect on cell membranes. This paper presents the results of the analysis of differential gene expression under the action of hexan-1-ol alcohol on a culture of cyanobacterium Synechocystis. Our data allowed us to identify groups of genes activated by the action of the alcohol fluidizer. These findings may be useful to select the best way to improve cyanobacterial resistance to alcohol toxicity. For the analysis of the alcohol-induced transcriptome, the control experiment was performed as follows: 20 mL aliquots from three 250 mL independent Synechocystis cultures grown under similar standard conditions were fixed by addition of 30 mL of pre-chilled to −20°C 1% phenol in 95% ethanol solution, mixed, centrifuged at 3,000 g for 3 min, decanted and used for the subsequent RNA isolation. For the alcohol stress treatment, an aliquot of hexan-1-ol was added up to 20 mM final concentration to the same three cultures used earlier for the control sample harvesting, vigorously mixed and incubated at the same growth conditions for 30 min. At the end of the treatment, 20 mL aliquots were taken from each hexanol-treated culture and fixed as described above. In total, six RNA isolation procedures according to https://bio-protocol.org/e1428 were performed during one experiment. rRNA quality was confirmed by gel electrophoresis. All three RNA samples from the control experiment were combined in equal amounts to obtain 1 control sample, and the same mixing was done for the hexan-1-ol treated samples. The overall experiment was repeated three times. Total Synechocystis RNA was treated with DNAse I (Thermo Fisher Scientific) and rRNA was depleted using bacterial RiboMinus Transcriptome Isolation Kit (Thermo Fisher Scientific). cDNA libraries for sequencing were prepared using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific), with 500 ng of rRNA depleted total RNA as starting material. Resulting whole transcriptome libraries were diluted to 100 pM concentrations and applied for template-positive Ion Sphere Particles generation using The Ion OneTouch System. Ion PGM System was initialized with Ion PGM Hi-Q View Sequencing Kit and two runs were performed in “300 + 300”-bp (700 + 700 flows) format using 318 Chip Kit v2. The whole transcriptome differential gene expression analysis was performed using DESeq2 package as a part of ReadXplorer v.2.2.3 pipeline. The resulting gene list for the transcriptome analysis consists of 1727 ORFs with well-defined P adjusted values. Expression of several hexan-1-ol inducible genes (hliB, ndhD2, lilA, sodB, clpB2, htpG, ocp and slr0095), selected based on table data, was confirmed using RT-qPCR. For all eight tested genes the induction of expression by the alcohol was confirmed.