strain: strain 168 tissue: entire bacterial cell growth phase: logarithmic growth treated with: 5 µg/ml gentamicin for 30 min
Treatment protocol
B. subtilis was treated at 37C aerobically with shaking with BSA (control, 100 µg/ml), or PGRP (human recombinant PGLYRP4, 100 µg/ml), or gentamicin (5 µg/ml) for 30 min, or with CCCP (carbonyl cyanide 3-chlorophenylhydrazone, 50 µM) for 15 min. Each experiment was repeated 3 times.
Growth protocol
B. subtilis (OD660 nm = 0.1) was incubated with control protein or antibacterial preparations in TRIS-Schaeffer medium with 0.05% NH4Cl, 10 µM ZnSO4, 0.2% glucose, and 2% LB, at 37oC aerobically with shaking.
Extracted molecule
total RNA
Extraction protocol
Total RNA was obtained from B. subtilis using Ambion RiboPureTM-bacteria RNA extraction kit after disrupting the cells by shaking with Zirconia beads according to the manufacturer’s instructions.
Label
biotin
Label protocol
The cDNA was synthesized with random hexamer primers, fragmented, and labeled with terminal transferase and biotin at the Genomic and RNA Profiling Core facility, Baylor College of Medicine, Houston, TX 77030, using the protocols provided by Affymetrix GeneChip® Technical Manual.
Hybridization protocol
Hybridization to the custom whole genome Affymetrix 900513 GeneChip B. subtilis Genome Array was done using Affymetrix Hybridization Oven 640 and Affymetrix GeneChip® Fluidics Station 450 and the protocols provided by Affymetrix GeneChip® Technical Manual at the Genomic and RNA Profiling Core facility, Baylor College of Medicine, Houston, TX 77030.
Scan protocol
Scanning, and data extraction was done using Affymetrix GeneChip® Scanner 3000 and the protocols provided by Affymetrix GeneChip® Technical Manual at the Genomic and RNA Profiling Core facility, Baylor College of Medicine, Houston, TX 77030.
The hybridization intensity data signals were analyzed, normalized, and corrected for batch effect using Affymetrix GeneChip® Command Console® Software (AGCC v.3.0). Signal average, noise average, scaling factor, % present, and % absent were calculated for each probe, from which the signal intensity of >78 was calculated as reliable expression. Using this cutoff, 3355 probes were classified as present out of total 5039 probes on the array. Signal intensities with all probes are shown in sample data table. The 'expressed_present_probes.txt (available on Series records) contains Signal intensities with present probes. Average signal intensities from 3 experiments were used to calculate fold increases in gene expression between treated and control groups, with signal intensity of 78 used as a minimum intensity, using the formula: average intensity in treated group/average intensity in control group (the 'Avg' columns). Transformed Ln (signal intensity) values, shown in columns 'Ln', were used for direct statistical comparisons of expression signals between treated and control groups by t-test (columns 't-test' ). The probes in the expressed_present_probes.txt are arranged in descending order from the highest to the lowest ratio of gene induction in PGRP/control (column 'Ratio' in 'expressed_present_probes.txt').
