Bacterial cells were cultured in liquid KB or hrp-inducing medium for 4 h or 8h at 28°C.
Extracted molecule
total RNA
Extraction protocol
Two ml of each bacterial suspension were centrifuged and total RNA was extracted from cell pellets using the Spectrum™ Plant Total RNA kit (Sigma-Aldrich, St. Louis, MO, USA). Total RNA quantity, integrity and purity were controlled using a Nanodrop 2000 instrument (Thermo Scientific, Wilmington, DE, USA) and a Bioanalyzer Chip RNA 7500 serie II (Agilent).
Label
Cy3
Label protocol
Cyanine-3 (Cy3)-labeled cRNA was prepared from 50 ng of RNA using the Low Input Quick Amp WT Labeling kit (Agilent Techonologies, Santa Clara, CA, USA) according to manufacturer's instructions for prokaryotic samples, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked using the NanoDrop 2000 instrument (Thermo Scientific, Wilmington, DE, USA).
Hybridization protocol
600 ng of Cy3-labelled cRNA (specific activity >15 pmol Cy3/ug cRNA) were fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following manufacturer's instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer were added to the fragmentation mixture and hybridized to Agilent-078853 8x60K custom microarray chip for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarray chips were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute at 37°C with GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned immediately after washing using the Agilent DNA Microarray Scanner with one color scan settings for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 um, Dye channel is set to Green and Green PMT is set to XDR Hi100%, XDR Lo 10%).
Description
Gene expression in Pto DC3000 cells grown 8h in KB medium, replicate 3
Data processing
The scanned images were analyzed with Feature Extraction Software 10.5.1 (Agilent) using default parameters (protocol GE1-105_Dec08 and Grid:078853_D_F_20151012) to substract the background and obtain spatially-detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
