Overnight culture of S. Typhimurium NBRC 12529 was harvested by centrifugation at 3,300 × g at 4°C for 15 min, followed by dilution in sterile water. ε-Polylysine (Chisso Corporation, Tokyo, Japan) was dissolved in water at 0.002% and filter-sterilized with EB-DISK 25 (pore size 0.2 μm, Kanto Chemical Co., Ltd., Tokyo, Japan). Five mL of 0.2% Bacto Soytone and 5 mL of 0.002% ε-Polylysine were added into test tubes, for the control sample, 5 mL sterile water was added instead of ε-Polylysine. Then, Salmonella cell suspension was added to the tubes and the optical density at 660 nm (OD660) of suspensions was adjusted to around 0.7, corresponding to 109 – 1010 CFU/ml. Finally, solutions in the tubes were poured into to petri-dishes and incubated at 30°C for 2 h.
Extracted molecule
total RNA
Extraction protocol
Salmonella cells attached on petri-dishes were scrubbed with spreading rod and mixed with the planktonic cells. The cell suspension was transferred into 50 mL conical centrifuge tube. The cells were collected by centrifugation at 5,800 × g for 10 min at room temperature. The quantity of RNA was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). RNA samples were further purified using RNase-free DNase I (Qiagen, Germany) and RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. Quality of the total RNA was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). RNA samples were stored at -80°C until further use.
Label
Cy3
Label protocol
Two-hundreds ng of purified total RNA were used to produce Cyanine 3-CTP dye labelled cRNA using the Quick Amp Labeling kit, and then purified with the RNeasy Mini Kit (Qiagen).
Hybridization protocol
For hybridization, 600 ng of labelled cRNA was fragmented in a 25 uL reaction mixture containing uL 1× Agilent fragmentation buffer and 10× 5 uL blocking agent at 60°C for 30 min. Afterwards, 25 uL of 2× GEx hybridization buffer HI-RPM was added to the fragmented cRNA and hybridized onto the arrays at 65°C for 17 h in Agilent hybridization oven with rotation (10 rpm). Then, microarrays were washed using gene expression wash buffer 1 and 2 for 1 min each, followed by a few seconds of air-dry.
Scan protocol
Slides were scanned on the Agilent DNA Microarray Scanner with Sureelect High-resolution Technology (SG11350602) using one color scan setting for 8 × 15k array slides (Scan resolution 5 um, Dye channel is set to Green and Extended Dynamic Range of 0.1 at 16-bit dynamic range 16-bit).
Description
Gene expression of Salmonella cells after 2h Polylysine treatment
Data processing
Raw microarray data were extracted from scanned images by using Agilent Feature Extraction (FE) software (version 10.7.3.1), and then exported to GeneSpring GX 13.0 version software (Agilent). Log2 fluorescence ratios were generated for each replicate spot and averaged. The expression fold change of a given gene was calculated as the ratio of the signal intensity of the test sample to the signal intensity of the control sample. Significance of these expression values was determined with P values of ≤0.05. The transcripts of predominantly upregulated or downregulated genes were identified using a ≥ 2- fold expression cutoff.
