strain: HSC5 genotype: Wild Type treatment: glucose biological replicate: 2
Growth protocol
Overnight cultures of the indicated Streptococcal strain were grown in Todd Hewitt broth +0.2% Yeast extract (ThyB), then in the morning back diluted 1:100 into the indicated C medium (either with 1% glucose or without) and allowed to grow at 37 degrees C in sealed culture tubes until an reaching an optical density (OD 600) of 0.2 (exponential phase of growth) or until increases in OD were not apparent after 30 minutes (approximately OD 600 of 1.2, stationary phase of growth)
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using glass beads (Lysing Matrix B, MP biomedicals) and a high-speed reciprocating shaking device (FastPrep FP-120, MP biomedicals). RNA was further purified using a commercially avaiable kit (Rneasy Mini kit, Qiagen) as per manufacturers instructions. Contaminating DNA was removed by treatment with DNaseI (Invitrogen) and samples were cleaned up using the RNeasy Mini kit. RNA was further analyzed by a fluorescent assay involving electrophoretic separation of RNA (Agilent 2100 Bioanalyzer) and rejected if the electropherogram (a diagram of fluorescence over time) showed degradation over time.
Label
cy3
Label protocol
First strand cDNA is generated by random primed reverse transcription (Superscript II; Invitrogen) utilizing the 3DNA Array 350MPX kit (Genisphere). For cDNA synthesis, 2x volume the RNA mass of random primer, and nuclease-free water is added to 4ug of total RNA for a total volume of 11ul. The solution is incubated at 80C for 10 minutes, then cooled on ice for 2 minutes. To each sample are added RNase inhibitor (Superase-In; Ambion) (1ul), 5X first strand buffer (4ul), dNTP mix (10mM each dATP, dCTP, dGTP, and dTTP) (1ul), 0.1M DTT (2ul), and Superscript II RNase H- Reverse Transcriptase (1ul). Reverse transcription is carried out at 42C for 2 hours. The reaction is terminated by adding 0.5M NaOH/ 50mM EDTA (3.5ul) and incubation at 65C for 15 minutes then neutralized with 1M Tris-HCL, pH 7.5 (5ul). The cDNA is cleaned using MinElute PCR Purification columns (Qiagen). Water is added (6.5ul) to the eluted cDNA (10ul), heated to 80C for 10 minutes, then cooled on ice for 2 minutes. The cDNA is poly-T tailed by adding 10x Tailing buffer (2.5ul), 10mM dTTP (4ul), and Terminal Deoxynucleotidyl Transferase (2ul). The reaction is incubated at 37C for 30 minutes. The reaction is terminated by heating to 95C for 10minutes, then cooled on ice for 2 minutes. Fluorophore/dendrimer specific oligos are then ligated to the appropriate cDNAs by adding 6X Ligation Mix (cy3 or cy5 specific) (5ul) and T4 DNA Ligase (2ul). The reactions are incubated at room temperature for 30 minutes. The ligation reaction is terminated by adding 0.5M EDTA and then vortexing for 10 seconds. The cDNA is again cleaned using MinElute PCR Purification columns (Qiagen). The cDNAs are quantitated via spectrophotometry to confirm recovery. Samples are paired and balanced by mass.
strain: HSC5 genotype: Wild Type treatment: no glucose biological replicate: 2
Growth protocol
Overnight cultures of the indicated Streptococcal strain were grown in Todd Hewitt broth +0.2% Yeast extract (ThyB), then in the morning back diluted 1:100 into the indicated C medium (either with 1% glucose or without) and allowed to grow at 37 degrees C in sealed culture tubes until an reaching an optical density (OD 600) of 0.2 (exponential phase of growth) or until increases in OD were not apparent after 30 minutes (approximately OD 600 of 1.2, stationary phase of growth)
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using glass beads (Lysing Matrix B, MP biomedicals) and a high-speed reciprocating shaking device (FastPrep FP-120, MP biomedicals). RNA was further purified using a commercially avaiable kit (Rneasy Mini kit, Qiagen) as per manufacturers instructions. Contaminating DNA was removed by treatment with DNaseI (Invitrogen) and samples were cleaned up using the RNeasy Mini kit. RNA was further analyzed by a fluorescent assay involving electrophoretic separation of RNA (Agilent 2100 Bioanalyzer) and rejected if the electropherogram (a diagram of fluorescence over time) showed degradation over time.
Label
cy5
Label protocol
First strand cDNA is generated by random primed reverse transcription (Superscript II; Invitrogen) utilizing the 3DNA Array 350MPX kit (Genisphere). For cDNA synthesis, 2x volume the RNA mass of random primer, and nuclease-free water is added to 4ug of total RNA for a total volume of 11ul. The solution is incubated at 80C for 10 minutes, then cooled on ice for 2 minutes. To each sample are added RNase inhibitor (Superase-In; Ambion) (1ul), 5X first strand buffer (4ul), dNTP mix (10mM each dATP, dCTP, dGTP, and dTTP) (1ul), 0.1M DTT (2ul), and Superscript II RNase H- Reverse Transcriptase (1ul). Reverse transcription is carried out at 42C for 2 hours. The reaction is terminated by adding 0.5M NaOH/ 50mM EDTA (3.5ul) and incubation at 65C for 15 minutes then neutralized with 1M Tris-HCL, pH 7.5 (5ul). The cDNA is cleaned using MinElute PCR Purification columns (Qiagen). Water is added (6.5ul) to the eluted cDNA (10ul), heated to 80C for 10 minutes, then cooled on ice for 2 minutes. The cDNA is poly-T tailed by adding 10x Tailing buffer (2.5ul), 10mM dTTP (4ul), and Terminal Deoxynucleotidyl Transferase (2ul). The reaction is incubated at 37C for 30 minutes. The reaction is terminated by heating to 95C for 10minutes, then cooled on ice for 2 minutes. Fluorophore/dendrimer specific oligos are then ligated to the appropriate cDNAs by adding 6X Ligation Mix (cy3 or cy5 specific) (5ul) and T4 DNA Ligase (2ul). The reactions are incubated at room temperature for 30 minutes. The ligation reaction is terminated by adding 0.5M EDTA and then vortexing for 10 seconds. The cDNA is again cleaned using MinElute PCR Purification columns (Qiagen). The cDNAs are quantitated via spectrophotometry to confirm recovery. Samples are paired and balanced by mass.
Hybridization protocol
Each sample pair (~14ul) is suspended in Formamide-based hybridization buffer (vial 7-Genisphere) (16ul), and Array 50dT blocker (Genisphere) (2ul). Two hybridizations are carried out in a sequential manner. The primary hybridization is performed by adding 30ul of sample to the microarray under a supported glass coverslip (Erie Scientific) at 43C for 16-20 hours at high humidity. Prior to the secondary hybridization, slides are gently submerged into 2X SSC, 0.2% SDS (at 62C) for 11 min., transferred to 2X SSC (at room temp.) for 11 min., transferred to 0.2X SSC (at room temp.) for 11 min., and then spun dry by centrifugation. Secondary hybridization is carried out using the Array 350 complimentary capture reagents (Genisphere). For each reaction, the following are added: 3DNA capture reagent with Cy3 (2.5ul), 3DNA capture reagent with Cy5 (2.5ul), SDS-based hybridization buffer (vial 6-Genisphere) (16ul), and Rnase/Dnase-free water (11ul). The secondary hybridization solution is incubated in the dark at 80C for 10 min. then 50C for 15 min. 'Hybridization is performed by adding 30ul secondary hybridization solution to the slide under a supported glass coverslip at 65C for 3 hr at high humidity in the dark. At hybridization termination, arrays are gently submerged into 2X SSC, 0.2% SDS (at 65C) for 11 min., transferred to 2X SSC (at room temp.) for 11 min., transferred to 0.2X SSC (at room temp.) for 11 min., and then spun dry by centrifugation. To prevent fluorophore degredation, the arrays are treated with Dyesaver (Genisphere).
Scan protocol
Slides are scanned on a Perkin Elmer ScanArray Express HT scanner to detect Cy3 and Cy5 fluorescence. Laser power is kept constant for Cy3/Cy5 scans and PMT is varied for each experiment based on optimal signal intensity with lowest possible background fluorescence. A low pmt setting scan is also performed to recover signal from saturated elements.
Data processing
Gridding and analysis of images is performed using ScanArray v3.0 (Perkin Elmer).
