Affymetrix GeneChip S. aureus Genome Array [S_aureus]

In vitro and in vivo models of Staphylococcus aureus endophthalmitis implicate specific nutrients in ocular infection.

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processedData

S. aureus SA564 and SA564-codY-mutant were grown in bovine aqueous humor, bovine vitreous humor and a chemically defined medium. Samples were extracted in midlog phase and affymetrix microarray processing was performed.

growth condition

In vitro and in vivo models of Staphylococcus aureus endophthalmitis implicate specific nutrients in ocular infection

S. aureus SA564 and SA564-codY-mutant were grown in bovine aqueous humor, bovine vitreous humor and a chemically defined medium. Samples were extracted in midlog phase and affymetrix microarray processing was performed.


P-MTAB-41249

S. aureus SA564 was inoculated to an OD 600nm of 0.02- 0.03 in filter-sterilized bovine aqueous humor, bovine vitreous humor and chemically defined medium. Microarray experiments were performed in duplicate for each condition. Cells were harvested for RNA isolation at mid exponential growth phase by mixing 1:1 with RNALater (Ambion).

P-MTAB-41250

Terminal Labeling Use Enzo BioArray Terminal Labeling Kit with Biotin-ddUTP to label the 3prim termini of the fragmented cDNA. Follow kit protocol.  * Prepare following mix Components Volume/amount 5X reaction buffer 12 ul 10X CoCl2 6 ul Biotin-ddUTP 1 ul  * Add 19 ul reaction mix to 40 ul fragmented cDNA  * Add 2 ul Terminal Deoxynucleotide Transferase  * Incubate reaction at 37C for 60 min  * Store the labeled products at -20C prior to hybridization

P-MTAB-41251

RNA Isolation Reagents and Materials - RNaseZap - 1.5 ml microcentrifuge tubes (RNase-free) - 0.5 ml microcentrifuge tubes (RNase-free) - Filter tips (20 ul, 200 ul, 1000 ul) - RNAlater - Versagene RNA Tissue Kit: Cat#VGR-0050FTD - RNase-free water - Lysozyme 10mg/ml (200ul lysozyme + 10ml TE buffer) - 100 % ETOH - DNase I-RQ (Invitrogen) - dNTPs (25mM) - Expand Long Template PCR kit - Northern Max-Gly Gel Prep/Running Buffer (Ambion) - 0.24-9.5 kb RNA Ladder (Life Technologies) - Glyoxal Sample Loading Dye (Ambion) - 37C waterbath RNA Prep Culture Prep   1. Overnight strain O/N.  2. Innoculate new culture OD600 = .01, and grow to mid log  3. Add equal volume RNAlater and store in 4C until use. RNA Extraction These steps should be completed without interruption to minimize variation between samples. Harvesting and lysis of cells is based on the enzymatic lysis and mechanical disruption protocol described in the RNA Protect Bacteria Reagent Handbook. Subsequent purification of RNA is based on the Total RNA Purification Kit (Versagene). Perform all steps at room temperature unless stated otherwise.  1. Remove a volume of cells corresponding to 3 X 104 - 5 X 107 cells/ml. Add an equal volume of RNA later, and vortex for 5 sec or invert tube several times. Can be stored at 4 until needed. 2. Follow protocol for isolation of total RNA from cultured cells. It is not necessary to perform the optional on-columm Dnase digestion (step 7). No protocol yet exists for gram negatives. Step 1.add 100ul lysozyme + TE buffer (-20) to sample and incubate at 25C for ~ 10 - 30min add 792 ul lysis solution + 8 ul TCEP (-20 standup). 8.2ul B-ME may also be used in place of TCEP Vortex, homogenize using syringe until no visible clumps KEEP LYSATE ON ICE!!!! Follow protocol beginning at step  2Step 11: elute with 50ul 2x using same eluate Spec samples if desired  Can store purified RNA at -80C DNase Digestion  1. Perform a 30 ul reaction to digest DNA. Incubate for 1 hr at 37 C. 20 ul sample (2 digestions/sample) 3 ul Buffer (rm temp) 3 ul DNase (Promega, yellow lid, stored at -20C) 4.0 ul RNase free H20 30 ul rxn  2. Use the RNA Clean-up Protocol (pg 15 step 2)in the Gentra kit to remove enzyme and buffer components. Elute 2x in 50 ul Rnase free H20.  3. Spec samples. Good range for RNA, A260/280 1.8-2.2 DNA Contamination PCR.   To detect small amounts of DNA contamination that would not be visible on an agarose gel, a fragment of the ribosomal protein gene, rplU, is amplified by PCR using the Expand Long Template system from Roche.  Make a dilution (200ng/ul)  1. Run a standard PCR  2. Analyze 5 ul of PCR product on a 1.2 % TAE-agarose gel. The expected product size is 300 bp. Agarose Gel Gel Buffer  Sample 0.6 g agarose 10 ml 50x TAE Buffer 5 ul sample 50 ml H2O 500 ml H20 1 ul dye 1 ml 50x TAE Buffer 1 ul ladder Quality Control/Agarose Gel Analysis For quality control, RNA samples are glyoxylated and analyzed by agarose gel electrophoresis using the Ambion NorthernMax-Gly system. Glyoxal contains DMSO and SDS, both protein denaturants. Glyoxalation helps to unfold RNA so that it will run down the gel.  1. Clean a gel box with RNAZap and rinse out with diH2O  2. Cast a 1% agarose gel using RNase-free agarose and 1x NorthernMax-Gly Gel Prep/Running Buffer (Ambion). Gel-make in a 50ml conical Running Buffer-rinse flask with RNAZap .5 g agarose- weigh in lid 270 ml H2O 45 ml diH2O 30 ml Buffer 5 ml Buffer  3. Glyoxylate 1 ul of RNA into 5 ul RNase-free H2O. Add 4 ul Glyoxal Sample Loading Dye (Ambion) to each sample and heat at 50 C for 30 min. OOPS! If you accidently add glyoxal to entire sample, dont heat it. Just clean it up using a kit. 1ul RNA 5 ul RNase-free H2O 4 ul Dye  4. Run gel at 5V/cm as measured between electrodes. Run at least 100ng of your sample. Gels do not need to be stained as the loading buffer contains EtBr. Run entire sample (10ul). No ladder is necessary.  5. The 23s and 16s rRNA should give clear bands with the 23s band showing twice the intensity. The tRNA and 5s rRNA bands should barely be visible as the RNeasy columns do not retain small RNA species below 200 bases. cDNA Synthesis and Labeling This assay utilizes reverse transcriptase and semi-random primers to produce DNA complementary to the RNA samples. The cDNA product is then fragmented by DNase I and labeled with biotinylated nucleotides at the 3prim termini mediated by terminal transferase.   Reagents and Materials * dNTP mix-10mM • Superscript II Reverse Transcriptase, Invitrogen Life Technologies, # 18064-071  * RNase Inhibitor, Ambion #2684 • NS random primer oligos, semi-random decamer, 75%G+C (stock is 3ug/ul)  * Spike control  * QIAquick PCR purification kit, Qiagen, #28104  * 10X One-Phor-All Buffer, Amersham Pharmacia Biotech, #27-0901-02  * DNase I, RNase-free, Roche Molecular Biochemicals, #776785  * Enzo Bioarray Terminal Labeling Kit, Affymetrix, #900181  * 1N NaOH solution, VWR #MK469360  * 1N HCl solution, VWR, #MK638860  * 100bp DNA ladder, Invitrogen, #10380-012  * GelStar nucleic acid gel stain, FMC, #50535  * 37C waterbath  * 98C heatblock A. cDNA synthesis The following protocol begins with 12 ug of size fractioned RNA. The incubation is performed in a thermocycler with RNase-Free PCR tubes.  * Prepare the following mix for primer annealing Components Volume or amount Final concentrations Fractioned RNA 12 ug 0.33 ug/ul (NS) primer (250ng/ul) 3 ul 25 ng/ul Spike Transcript 2 ul DI H20 Up to 30 ul.  * Anneal program (70C for 10 min, 25C for 10 min, hold at 4C).  * Prepare the following mix for cDNA synthesis. 21 ul of mix are added to each sample. Components Volume/amount Final concentration 5X 1st strand buffer 12 ul 1X 100 mM DTT 6 ul 10 mM 10 mM dNTPs 3 ul 0.5 mM  * Add components individually, always keep enzymes on ice RNaseIN (40U/ul) 1.5 ul 1.0 U/ul SuperScript II (200U/ul) 7.5 ul 25 U/ul  * cDNA program (25C for 10 min, 37C for 60 min, and 42C for 60 min; inactivate enzyme at 70C for 10 min and hold at 4C). We usually let this reaction go overnight.  B. Removal of RNA * Transfer reactions to 1.7 ml microfuge tubes  *Add 20 ul of 1N NaOH and incubate at 65C for 30 min  * Add 20 ul of 1 N HCl for the titration, add H2O if necessary for 100 ul total volume. C. Purification and quantitation of cDNA synthesis products  * Use QIAquick PCR purification kit to clean up reaction (not minielute). To ensure all ETOH is removed, spin column 10 min following wash step, may need to blot with tissue.  * Elute cDNA 2x with 30 ul elution buffer for a total volume of 60 ul.  * Speed vac the sample for 10 min at 55C to remove ETOH.  * Quantitate cDNA at 260nm (1.0 A260 unit = 33 ug/ml of single stranded cDNA).  * Save 3.5 ul for analysis by agarose gel in step E. Note: Typical cDNA yields are 4-6 ug/60ul of eluted product. D. cDNA fragmentation Components Volume/amount Concentration 10X One-Phor-All buffer 5 ul 1X cDNA 3 ug - DI H20 Bring total volume to 50 ul DNase I (Roche) stock 10 U/ul 3 ul 0.06 U/ug of cDNA Note: Use 1X One-Phor-All buffer for the dilution of DNase I from stock solution prior to the fragmentation reaction. 1X Buffer  * 270 ul dH20  * 30 ul One-Phor-All Buffer DNase I* (working concentration)  * 1.5 ul Roche DNase  * 248.5 ul 1X One-Phor-All buffer  * Add 1 ul per ug cDNA (3ul as stated above)  * Add diluted DNase last, mix, immediately begin reaction. Very Time Sensitive!!!!! Use a timer!!!  * Incubate the reaction in 37C for 10 min, then inactivate enzyme at 98-100C for 10 min. E. Quality Control/Agarose gel electrophoresis For quality control pre- and post-fragmented cDNA samples are analyzed by agarose gel electrophoresis  * Cast a 2% agarose gel using GelStar nucleic acid gel stain at a dilution of 1:10000 using either TAE of TBE buffer. 1g agarose 50 ml diH20 5ul GelStar stain 1 ml TAE  * Analyze 5ul (~500ng) of fragmented cDNA and 3.5ul unfragmented cDNA (~500ng) as a control  * Use 2.5ul (250ng) of Ready-load 100 bp DNA ladder as marker for size determination. The desired fragment size should be distributed within 50-200 bases. F. Terminal Labeling Use Enzo BioArray Terminal Labeling Kit with Biotin-ddUTP to label the 3prim termini of the fragmented cDNA. Follow kit protocol.  * Prepare following mix Components Volume/amount 5X reaction buffer 12 ul 10X CoCl2 6 ul Biotin-ddUTP 1 ul  * Add 19 ul reaction mix to 40 ul fragmented cDNA  * Add 2 ul Terminal Deoxynucleotide Transferase  * Incubate reaction at 37C for 60 min  * Store the labeled products at -20C

P-MTAB-41252

Title: Affymetrix CHP Analysis (ExpressionStat)

P-MTAB-41253

Page 9 of 22 Hybridization Hybridization mix preparation 1. Take probe arrays out and equilibrate them to room temperature, to keep the septa pliable. In general, avoid touching the glass surface of the chip to avoid smears, scratches. 2. Turn rotisserie on; set to 45°C. It will take only a few minutes to warm up. 3. Thaw all reagents for the cocktail completely, mix well and spin down before use, especially the hybridization control cRNA mixture. The 2X MES hybridization buffer is kept at 4°C, protected from light. 4. Make cocktail (this recipe already incorporates extra volume per array for waste/loss): per Standard array final conc. Control Oligonucleotide B2 (3 nM, Affymetrix) 5 μL 50 pM 20X Eukaryotic Hybridization Controls (Affymetrix) 15 μL varies Herring Sperm DNA (10 mg/mL, Promega) 3 μL 0.1 mg/mL Acetylated BSA (50 mg/mL, Invitrogen) 3 μL 0.5 mg/mL 2X Hybridization buffer 150 μL 1X Fragmented cRNA (15 μg) 0.05 μg/μL H20 plus cRNA above 124 μL to final volume of: 300 μL 5. Heat the hybridization cocktail for 5 min @ 99°C. 6. Meanwhile, wet the array: Chip prehybridization 1. Prepare 1X hybridization buffer; approximately 200 μL per standard probe array. 2. With a slightly beveled pipet tip, pierce the upper right septum to vent. 3. With a pipetman, fill another tip with 200 μL 1X Hybridization buffer. 4. Pierce the other septum carefully and fill the array, keeping the tip perpendicular to the chip/septa. 5. Remove both pipet tips, and make sure that the bubble is moving easily across the array. 6. Place arrays in the rotisserie holder; snap into slots (check that both sides are firmly snapped in). Load arrays in a balanced configuration (with both holders on the same side) to avoid stress on the rotisserie motor. 7. Rotate the filled arrays at 60 rpm to prehybridize for 10 min @ 45°C. Hybridize sample 1. Transfer the hybridization cocktail from 99°C to 45°C and heat for 5 min. 2. Spin heated cocktail at max speed for 5 min to precipitate insoluble materials. 3. Remove the arrays from the rotisserie. 4. To remove prehyb buffer from array, simply reverse the loading sequence above. 5. Pierce upper septum as before to vent. 6. Depress the pipetman plunger, carefully pierce the other septum and remove the solution. Affy protocol050404 Page 10 of 22 7. Then, fill the array with 200 μL of the clarified hybridization cocktail; avoid any insoluble material at the bottom of the tube! The remaining cocktail can be saved and stored at -20°C. 8. Once again, check that the bubble is moving freely across the array. 9. Septa can be covered with Tough Spots (Diversified Biotech) to prevent evaporation/leaking. 10. Rotate the arrays at 60 rpm @ 45°C for 16 hours.

In vitro and in vivo models of Staphylococcus aureus endophthalmitis implicate specific nutrients in ocular infection

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