The strain was grown in Todd-Hewitt broth to A600=0.7
Extracted molecule
total RNA
Extraction protocol
RNA was isolated using RNeasy Mini Kit (QIAGEN, Valencia, CA). cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, Carsbad, CA)
Label
Biotin-N6-ddATP
Label protocol
The 3' terminus of the fragmented cDNA was labeled with Biotin-N6-ddATP (ENZO, New York, NY) at 37°C for 2 h using 50 U of terminal deoxynucleotide transferase (New England Biolabs, Ipswich, MA)
Hybridization protocol
The GeneChips were hybridized with 1.86 ug of biotin end-terminus labeled cDNA in 1X hybridization buffer [100 mM Mes, 1 M NaCl, 20 mM EDTA, and 0.01% Tween 20 (Pierce Chemical, Rockford, IL), 50 pM control oligonucleotide B2 (Affymetrix), 20 ug herring sperm DNA (Promega, Madison, WI), 100 ug acetylated bovine serum albumin (Invitrogen)] for 16 h at 45°C on a rotisserie at 60 r.p.m. Following hybridization, the hybridization cocktail was removed and the GeneChip filled with non-stringent buffer A [0.9 M sodium chloride, 60 mM sodium phosphate, 6 mM EDTA, and 0.01% Tween 20]. GeneChips were post-processed on the automated Affymetrix GeneChip Fluidics Station 450 using the following protocol: Wash 1: 10 cycles of 2 mixes/cycle with non-stringent buffer A at 25°C; Wash 2: 4 cycles of 15 mixes/cycles with stringent buffer B [100 mM Mes, 0.1 M NaCl, and 0.01% Tween 20] at 45°C; First stain: stain probe array for 10 min at 25°C in streptavidin-phycoerythrin (SAPE) solution [1x Mes stain buffer (100 mM Mes, 1 M NaCl, and 0.05% Tween 20), 2 mg/ml acetylated BSA, and 10 ug/ml SAPE (Molecular Probes, Eugene, OR)] post stain: wash 10 cycles of 4 mixes/cycle with non-stringent buffer A at 300C; second stain: stain probe array for 10 min in antibody solution [1x Mes stain buffer, 2 mg/ml acetylated BSA, 0.1 mg/ml normal goat IgG (Sigma-Aldrich, St. Louis, MO), and 3 ug/ml biotinylated antibody (Vector Laboratories, Burlingame, CA); third stain: stain probe array for 10 min in SAPE solution at 25°C; final wash: 15 cycles of 4 mixes/cycle with non-stringent buffer A at 30°C.
Scan protocol
The probed arrays were scanned at 570 nm at a resolution of 1.56 µm using a confocal GC3000 laser scanner (Affymetrix)
Description
End-terminus biotin-labeled cDNA (1.86 µg) was hybridized to S. pyogenes GeneChip genome arrays following the prokaryotic hybridization protocol recommended in the Affymetrix GeneChip Expression Analysis Technical Manual available at the Website http://www.affymetrix.com/support/technical/manual/expressionmanual.affx.
Data processing
Gene expression levels were calculated with GeneSpring 7 software (Silicon Genetics, Redwood City, CA)
