WT and three non-adahesive mutants (#1476, #1543, and #1649) growing exponentially in MRS or m-ILS medium were sampled at 3 hours after 5%(v/v) inoculation.
Extracted molecule
total RNA
Extraction protocol
A volume of bacterial culture in exponential growth phase was mixed with twice the volume of RNA Protect Bacteria Reagent (Qiagen) and centrifuged (20,000 g, 5 min, 4°C). The bacterial pellet was suspended in 1.0 mL of Tris–EDTA buffer (Tris, 50 mM Tris,; 10 mM EDTA, 10 mM; (pH 8.0), that contained 10% (w/v) of sucrose.; thereafter, Llysozyme (50 mg/mL , 50 μL;) (Sigma-Aldrich) was added to break the cell walls. Tand the suspension was incubated for 10 min at 37°C. The enzyme-treated suspensionsand were centrifuged (20,000 g, 5 min, 4°C). RNA was extracted from the precipitated spheroplasts with RiboPure-Bacteria (Thermo Fisher Scientific).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the Low Input Quick Amp WT Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop Spectrophotometer.
Hybridization protocol
600 ng of Cy3-labelled cRNA (specific activity >15.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2× Hi-RPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent Custom Microarrays for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G4900DA) using one color scan setting for 8x15k array slides.
Data processing
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1_1200_Jun14) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
