Oligo acgh wash buffer 2

Whole-genome M. tuberculosis microarray slides were purchased from Agilent Technologies through the Bacterial Microarray Group at St. George’s (BμG@S), University of London. For cDNA synthesis, 2 μg wild-type H37Rv and ΔsfdS knockout isolated from 24-h starved cultures was used. The cDNA was labeled individually with both Cy-3 and Cy-5 dyes (GE Healthcare) using Superscript III reverse transcriptase (Invitrogen). Dye swaps were performed, and the cDNA was hybridized to an 8 Chamber Agilent slide at 65°C for 16 h before slide was washed with Oligo aCGH wash buffer 1 (Agilent) for 5 min at room temperature and Oligo aCGH wash buffer 2 (Agilent) for 1 min at 37°C. Slides were stabilized using Agilent stabilization and drying solution according to the manufacturer’s instructions.
Slides were scanned at 5 μm using an Agilent Technologies microarray scanner at BμG@S. Txt files created by the Agilent scanner were analyzed using Genespring 14.5 filtering on flags and expression. t test against zero was performed using a P value of <0.05 with Benjamini-Hochberg multiple testing correction and 2-fold cutoff. Array design is available in ArrayExpress (accession no. A-BUGS-41). Microarray data have been deposited into ArrayExpress (accession number E-MTAB-9327).

cDNA preparations that were positive for S. suis orf2 by qPCR were used for microarray experiments. cDNA (0.5–2.5 µg) was labeled with cyanine 3-d-UTP using the Genomic DNA Enzymatic Labeling kit (Agilent Technologies) according to manufacturer’s instructions and purified using Amicon Ultra-30K membrane filters (Millipore). Prior to hybridization, pure labeled cDNA in Agilent blocking reagent and Agilent Hi-RPM buffer was incubated for 3 min at 95°C followed by 30 min at 37°C. The mixture was then hybridized to one array of an 8 × 15 k custom S. suis oligo-array (Agilent Technologies) containing probes based on genome sequences of strain P1/7 [27] and probes designed on incomplete genome sequences of strains 891,591, 8067, 7917, and 6388 and incubated at 65°C in a rotator hybridization oven. After 17 h, arrays were washed for 5 min at RT in Oligo aCGH wash buffer 1 (Agilent Technologies) and subsequently for 1 min at 37°C in Oligo aCGH wash buffer 2. Arrays were dried at RT and scanned using a Surescan high-resolution DNA microarray scanner (Agilent Technologies), followed by data analysis using Feature extraction software and Genespring GX software, both also from Agilent Technologies.