# Adaptation of the Marine Bacterium _Shewanella baltica_ to Low Temperature Stress
## Materials and Methods
### RT-qPCR Assay and Data Analysis  
Synthesis of cDNA was performed in a total volume of 20 \(\upmu\)L; the reaction contained 1 \(\upmu\)g of total RNA, 4 \(\upmu\)M random hexamers (Invitrogen by Thermo Fisher Scientific, Carlsbad, CA, USA), 1 mM dNTP Mix (Invitrogen by Thermo Fisher Scientific, Carlsbad, CA, USA), 1X RT buffer, 5 mM DTT, 1.25 U of RNase Inhibitor and 0.5 U of AMV Reverse Transcriptase Native (EurX, Gdansk, Poland), according to the manufacturer's instructions. Priming of RNA-hexamers-dNTP was performed at 65 \({}^{\circ}\)C for 5 min and then was chilled on ice. Reverse transcription was performed in a thermocyler under the following conditions: 20 \({}^{\circ}\)C for 10 min (random primer extension), 50 \({}^{\circ}\)C for 50 min (cDNA synthesis) and 75 \({}^{\circ}\)C for 10 min (inactivation). Reverse transcriptions were performed in duplicate. Samples of cDNA were diluted 1:20 in water, and 4 \(\upmu\)L aliquots were used as a template in subsequent qPCR reactions.  
Primers for qPCR of target and reference genes were designed using the Clone Manager 10 software (Sci Ed Software LLC, Westminster, CO, USA) and synthesized by Sigma-Aldrich (St. Louis, MO, USA) custom service; primer sequences are available in Supplementary Table S4. All PCR products obtained with the designed primers were checked for consistency with the expected sequence by [PERSON] sequencing (Macrogen Europe, Amsterdam, Netherlands). Real-time qPCR amplification was performed in a total volume of 10 \(\upmu\)L using the SG qPCR Master Mix (2X) kit (EurX, Gdansk, Poland) according to the manufacturer's instruction, with the 0.5 \(\upmu\)M final concentration of each primer. Reactions were run in the LightCycler(r) 480 Instrument II (Roche Diagnostics Ltd., Rotkreuz, Switzerland) at the following conditions: 95 \({}^{\circ}\)C for 2 min (initial denaturation), followed by 35-45 cycles of 94 \({}^{\circ}\)C for 15 \(\upmu\)S, 53 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 30 s. Melting curve analysis was performed between 65 \({}^{\circ}\)C and 95 \({}^{\circ}\)C. Crossing threshold (Ct) values were generated by the LightCycler(r) 480 Software version 1.5 (Roche Diagnostics GmbH, Rotkreuz, Switzerland). We assumed that the optimal Ct values should be in the range of 20-32 cycles; if lower or higher Ct values were obtained, volumes of cDNA template were adjusted and qPCR amplifications were repeated. Each cDNA template was amplified twice with two technical replicates for each assay. Negative controls, such as reactions without reverse transcriptase or without cDNA template, were included in our analysis. For validation of reference genes, we used a set of 10-fold dilutions (ranging from 1 to 1:10,000) of cDNA as a template for qPCR. The stability and validity of five candidate reference genes for normalization of RT-qPCR results were determined by the BestKeeper version 1 tool [101] using raw Ct values. The 2-\({}^{\Delta\Delta\text{Ct}}\) algorithm was used to calculate the relative gene expression [102]. All results were checked for outliers with [PERSON] and [PERSON]'s two sided test for multiple outliers [103]; outlier criterion was set at the modified Z-score \(\geq\) 3.5.