Investigation Title Comparative genomic hybridization of forty clinical isolates from twelve common serovars within Salmonella enterica subspecies I (sspI) to identify the conserved gene pool present Comment[Submitted Name] Identification of Core and Varying Components of the Salmonella enterica subspecies I Genome Using Microarray Experimental Design comparative_genome_hybridization_design strain_or_line_design comparative genomic hybridization by array Experimental Design Term Source REF mo mo EFO Comment[ArrayExpressReleaseDate] 2005-12-06 Comment[AEMIAMESCORE] 4 Comment[ArrayExpressAccession] E-SGRP-1 Comment[MAGETAB TimeStamp_Version] 2010-09-06 23:06:01 Last Changed Rev: 13833 Experimental Factor Name Serovar Experimental Factor Type strain_or_line Experimental Factor Term Source REF Person Last Name Dougan Marooney Qureshi Baker Ivens Woodward Fookes Anjum Person First Name Gordon Chris Matloob Stephen Al Martin Maria Muna Person Mid Initials J. F. Person Email Person Phone Person Fax Person Address Person Affiliation Wellcome Trust Sanger Institute Person Roles submitter Person Roles Term Source REF Quality Control Type technical_replicate peer_review_quality_control Quality Control Term Source REF mo Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2005-12-06 PubMed ID 16299280 Publication DOI 16299280 Publication Author List Anjum, Muna F.; Marooney, Chris; Fookes, Maria; Baker, Stephen; Dougan, Gordon; Ivens, Al; Woodward, Martin J. Publication Title Identification of Core and Variable Components of the Salmonella enterica Subspecies I Genome by Microarray Publication Status journal_article Publication Status Term Source REF Experiment Description We have performed microarray hybridization studies on forty clinical isolates from twelve common serovars within Salmonella enterica subspecies I (sspI) to identify the conserved gene pool present. Protocol Name P-MEXP-9324 P-MEXP-9325 P-MEXP-9326 P-MEXP-9327 P-MEXP-9338 Protocol Type grow compound nucleic_acid_extraction labeling hybridization Protocol Description For preparation of genomic DNA, cells were grown overnight in LB broth at 37C The bacterial strains used in this study include clinical and field isolates of Salmonella from diseased and healthy animals or patients, and the sequenced S. Typhi (CT18) and S. Typhimurium (LT2) laboratory strains. The resistance of most animal isolates to the following compounds was known: Ciprofloxacin, Naladixic acid, Tetracylin, Chloramphenicol, Trimethoprim, Kanamycin, Streptomycin, Hexane, Cyclohexane. Strains that were resistant to three or more antibiotcs were termed Multiple Drug Resistant (MDR) and that showing resistance to cyclohexane and antibiotics were termed multiple antibiotic resistance (MAR). The breakpoints defining resistance are detailed in Randall et al. For preparation of genomic DNA, cells were grown overnight in LB broth at 37C and DNA was isolated using QIAGEN DNeasy Tissue Kit (no.69504, QIAGEN) or the CTAB (hexadecyltrimethylammonium bromide, Sigma-Aldrich Ltd) method. Cy Labelling

Notes: - Always use gloves, separate solutions and special tubes/tips when working with RNA!
- Protect Cy dyes from light exposure as much as possible!

1. Set temperature blocks to 100 C and 37 C (or waterbath). Place all required reagents and samples on ice (experimental and reference gDNA random primer, Superscript enzyme, buffer and Cy dyes).
2. Mix 21 ul of gdna solution with 20 ul of 2.5X random primer/reaction buffer mix from BioPrime kit (Gibco/BRL)
3. Incubate the reaction mixtures at 100 C for 5 min. Place Cy dye on ice for step 6.
4. Snap-chill the tubes on ice. Spin down reaction mixtures for 15 sec and place the tubes on ice.
5. On ice add 5 ul of 10X dNTP mix (1.2 mM each dATP, dGTP and dTTP and 0.6mM dCTP in 10mM tris 8.0, 1mM EDTA)
6. Add 3 microlitres of dCTP Cy-labelled nucleotide to each tube. Remember which dye you use for experiment vs reference to bear in mind the forward/reverse dye swap, if any. Change tips between tubes. Keep exposure to light of Cy dyes to minimum.
7. Add 1 microlitre of Klenow enzyme from the BioPrime kit taken freshly from freezer. Make sure that there is no drop outside of tip after taking enzyme. Change tips between tubes. When enzyme gets low, spin down briefly before use. Put enzyme back to freezer immediately after use.
8. The total volume of the reaction mixture should now be 50 microlitres. Vortex reactions and spin down for 15 sec.
9. Incubate the reactions at 37 C for 2 h. Cover with lid for light protection.
10. Add 5 ul of BioPrime Kit stop buffer.
11. Prepare AutoSeq G-50 columns (Amersham), 1 to 2 columns for each labeling reaction:
- Resuspend the resin in the column by vortexing gently.
- Loosen the cap 1/4th turn and snap off the bottom closure.
- Place the column in a 1.5 ml screw-cap microcentrifuge tube for support. Alternatively, cut the cap from a flip-cap tube and use this tube for support.
- Spin the column for 1 min at 2000 x g. Start the timer and the centrifuge simultaneously. Use the column immediately after preparation to avoid drying of the matrix.
12. Purify the labeled cDNA as follows:
- Place the column in a new 1.5 ml tube and slowly apply the sample to the centre of the angled surface of the compacted resin bed, being careful not to disturb the resin. Do not allow any of the liquid to flow around the sides of the bed.
- Spin the column for 1 min at 2000 x g. Start the timer and microcentrifuge simultaneously. The purified sample is collected at the bottom of the tube. Discard the column.
13. Pool purified experimental cDNAs with corresponding reference cDNAs
14. Add 1/10th volume of 3M NaAc pH 5.2 (11 microlitres) and 3 volumes of RT 100% EtOH (363 microlitres), mix, and precipitate at RT or -70 C for 20 min. (or -20 C overnight).
15. Centrifuge at RT for 10 min at 14,000 rpm. Pellet should appear purple. Discard SN, add 100 microlitres of 70% EtOH (4C), mix gently by tipping with finger, and spin for 5 min (same tube orientation!). Aspirate most SN, spin 5 sec, and remove rest of liquid with pipette.
16. Air dry for 5 min at RT. Make a master mix for all your reactions (calculate some extra, e.g. for reaction): ~30 microlitres/reaction of hybridization buffer (5 x SSC, 6 x Denhardts 60 mM TrisHCl pH 7.6, 0.12% sarkosyl, 48% formamide; filter sterilized) and 2 microlitres to each reaction of tRNA from yeast, Sigma R8508) and 4 microlitres of DDW. Add to each ss cDNA pellet and resuspend pellet with pipette.
17. Place hybridization mixtures in 100 C temperature block for 5 min. Let the tubes cool down at RT for 10 min. Spin the tubes for 15 sec to remove evaporated liquid from lids. Gently vortex. Hybridisation

Numbers continue from Labelling procedure.

18. Immediately before use, clean microarrays and coverslips with dust gun. Add hybridization mixture onto middle of inverted clean 60x25 mm LifterSlip. Slowly lower a labeled microarray with the DNA side onto the LifterSlip to prevent bubbles and misplacement.
19. Prepare Genetix chamber with one rectangule shape Whatman filter paper, and add 1.5 ml of 15 x SSC to each filter. Put microarrays into chamber and incubate at 49 C for ~16 hrs in hybridization oven.
20. Remove microarrays from humid chamber and immediately place into a staining jar filled with wash solution 1 (2 x SSC, filter sterilized. Let coverslip fall off by itself (~15 sec).
22. Put microarrays in slide rack. Wash in a staining jar with ~100 ml of solution 1 at RT for 5 min with gentle shaking.
22. Transfer microarrays in slide rack to wash solution 2 (0.1 x SSC, 0.1% SDS, filter sterilized). Wash at RT for 15 min with gentle shaking as before.
23. Repeat step 23.
24. Transfer microarrays in slide rack to wash solution 3 (0.1 x SSC, filter sterilized). Wash at RT for 15 min with gentle shaking as before.
25. Exchange wash solution 3 once to get rid of all SDS.
26. Quickly transfer microarrays in slide rack to a centrifuge and spin at 1200 rpm for 3 min to dry the slides (Make sure racks sit on clean filter paper over clean surface)
27. Store microarrays in light protected slide box at RT. Scan as soon as possible. Protocol Parameters Growth medium;Temperature;Time (min);Time (max); Amount of nucleic acid used; Chamber type;Quantity of labeled target used;Time;Volume;Temperature; Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology SDRF File E-SGRP-1.sdrf.txt Term Source Name mo ArrayExpress mo EFO The MGED Ontology Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version