Investigation Title Transcription profiling of N. gonorrhoeae wild type strain grown in anaerobic conditions with sodium nitrate supplied in media compared to those grown in capnoaerobic conditions Comment[Submitted Name] N. gonorrhoeae anaerobic - sodium nitrite supplied within the media Experimental Design co-expression_design replicate_design growth_condition_design transcription profiling by array Experimental Design Term Source REF mo mo mo EFO Comment[AEMIAMESCORE] 5 Comment[SecondaryAccession] Comment[ArrayExpressReleaseDate] 2011-05-01 Comment[ArrayExpressAccession] E-MEXP-1660 Comment[MAGETAB TimeStamp_Version] 2010-08-09 22:59:01 Last Changed Rev: 13058 Experimental Factor Name ATMOSPHERE Experimental Factor Type growth_condition Experimental Factor Term Source REF Person Last Name Saunders Person First Name Nigel Person Mid Initials J Person Email nigel.saunders@path.ox.ac.uk Person Phone 01865 275521 Person Fax 01865 275515 Person Address South Parks Road, Oxford, Oxfordshire, OX! 3RE, United Kingdom Person Affiliation University of Oxford Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type biological_replicate Quality Control Term Source REF The MGED Ontology Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2011-05-01 PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Experiment Description Transcriptional profiling of N. gonorrhoeae grown in anaerobic conditions (with sodium nitrite supplied by adding 20% w/v solution to a final concentration of 5mM) compared to a control grown in capnoaerobic conditions (normal atmosphere +5% CO2) Protocol Name P-MTAB-843 P-MTAB-848 P-MTAB-842 P-MTAB-847 P-MTAB-849 P-MTAB-846 P-MTAB-844 Protocol Type grow grow nucleic_acid_extraction labeling labeling hybridization bioassay_data_transformation Protocol Description Anaerobic bacterial cultures were grown on GC Agar (Difco Laboratories) containing the Kellogg supplements, 5 mg/liter ferric nitrate (Kellogg et al., 1963) and 5mM NaNO2 overnight at 37 °C in a 2.5 L anaerobic jar with AnaeroGen catalyst packs (Oxoid) until colonies were 1-2mm in diameter.
(Parameters: start time = 2, stop time = 3, time unit = days, min temperature = 37, temperature unit = C, media = GC media) Capnoaerobic bacterial cultures were grown on GC Agar (Difco Laboratories) containing the Kellogg supplements and 5 mg/liter ferric nitrate (Kellogg et al., 1963) overnight at 37 °C in air supplemented with 5% CO2.
(Parameters: start time = 14, stop time = 18, time unit = hours, min temperature = 37, temperature unit = C, media = GC media ) Bacteria are either looped off solid media plates and suspended in 500ul RNAlater



or



For liquid cultures add 2x culture volume RNAlater mix the culture well with the RNAlater and spin out the pellet of cells. Add 500 ul of RNAlater to the pellet and resuspend the cells in the RNAlater.



Spin the RNAlater bacterial suspensions at 5000 rpm for 15 minutes.



Pipette off the RNAlater, leaving a bacterial pellet.



Add 1 ml of TRIzol or TRI Reagent to the pellets.



Vortex the pellet in the TRIzol Reagent for 10 minutes, making sure that the pellet is well mixed into the TRIzol.



Leave the tubes at room temperature for 10 minutes or more.



Add 300 ul of chloroform to the TRIzol tubes and shake to mix for 15 seconds, then incubate at room temperature 2-3 minutes.



Spin these tubes at 13,000 rpm for 15 minutes in the pre-chilled 4°C microfuge.



Take off the aqueous phase (>600 ul)



Add 800 ul isopropanol (optional and 2 ul GlycoBlue). Shake gently.



Leave tubes at room temperature for 10 minutes.



Spin tubes at ≥ 13,000 rpm for 15 minutes in the pre-chilled 4°C microfuge.



Take off isopropanol supernatant



Add 100 ul of RNase-free water to the pellet



The RNA is then immediately cleaned up using Qiagen RNEasy mini kit as per manufacturer’s instructions



Add 1 ul superaseIN to the 30 ul elution of RNA and aliquot RNA for freezing in -80 freezer and quality control on Agilent Bioanalyzer.






(Parameters: Extracted product = total_RNA, Amplification = none) Day 1:

In a 0.2 ml PCR tube, 20 μg of RNA was made up with RNAse-free water to 13.4 μl volume. 2 μl of random primers (3 μg/μl; Invitrogen) was added to it, then heated at 70°C for 10 min and immediately snap-chilled on ice. To the same tube, was added 6 μl of 5X first strand buffer (Invitrogen), 3 μl of 0.1 M DTT (Invitrogen), 0.6 μl of dNTP mix (25 mM dATP, dTTP, dGTP, and 10 mM dCTP; Invitrogen), 3 μl of either dCTP-Cy3 or dCTP-Cy5 (Amersham), and 2 μl of Superscript III (Invitrogen) to a final reaction volume of 30 μl. The labelling reaction was incubated at 42°C in an iCycler (BioRad) for 2 h. At the end of 2 h, 1.5 μl of 1 M NaOH was added to the reaction tube and incubated at 70°C for 20 min to hydrolyze the RNA and stop the labelling reaction. Then 1.5 μl of 1M HCl was added to the tube to neutralize the mixture.



The labelled mixture was cleaned up using a DNA Clean & Concentrator-25 column (Genetix) according to manufacturer’s instruction, except for one modification, where after the first loading of labelled reaction and centrifugation, the eluate was re-loaded onto the same column and re-centrifuged to maximize yield recovery. The final elution volume is 35 μl for each labelled cDNA.
(Parameters: Amount of nucleic acid labeled = 20, Label used = Cy5, Amplification = none, Mass unit = Micro gram) Day 1:

In a 0.2 ml PCR tube, 20 μg of RNA was made up with RNAse-free water to 13.4 μl volume. 2 μl of random primers (3 μg/μl; Invitrogen) was added to it, then heated at 70°C for 10 min and immediately snap-chilled on ice. To the same tube, was added 6 μl of 5X first strand buffer (Invitrogen), 3 μl of 0.1 M DTT (Invitrogen), 0.6 μl of dNTP mix (25 mM dATP, dTTP, dGTP, and 10 mM dCTP; Invitrogen), 3 μl of either dCTP-Cy3 or dCTP-Cy5 (Amersham), and 2 μl of Superscript III (Invitrogen) to a final reaction volume of 30 μl. The labelling reaction was incubated at 42°C in an iCycler (BioRad) for 2 h. At the end of 2 h, 1.5 μl of 1 M NaOH was added to the reaction tube and incubated at 70°C for 20 min to hydrolyze the RNA and stop the labelling reaction. Then 1.5 μl of 1M HCl was added to the tube to neutralize the mixture.



The labelled mixture was cleaned up using a DNA Clean & Concentrator-25 column (Genetix) according to manufacturer’s instruction, except for one modification, where after the first loading of labelled reaction and centrifugation, the eluate was re-loaded onto the same column and re-centrifuged to maximize yield recovery. The final elution volume is 35 μl for each labelled cDNA.


(Parameters: Amount of nucleic acid labeled = 20, Label used = Cy3, Amplification = none, Mass unit = Micro gram) Hybridization sample preparation:

A pair of cleaned up Cy-3 and Cy-5 labelled cDNAs was combined and placed in a Concentrator 5301 speed vacuum (Eppendorf; Histon, Cambridge) and the volume was evaporated down to 30 μl using function 2 setting for evaporation of aqueous solutions with no heating. 30 µl of 2X genHYB (Genetix; New Milton, Hampshire) was added to this and mixed by pipetting up and down, then transferred into a clean 0.2 ml PCR tube which was then pre-warmed to 42°C in an iCycler. Determination of Cy-dye incorporation efficiency on NanoDrop spectrophotometer (LabTech International Ltd.; Lewes, East Sussex) was done according to manufacturer’s instructions.



Prehybridization:

The prehybridization solution was made up by mixing 5 ml of 100 mg/ml BSA, 8.75 ml of 20X SSC (Sigma; Gillingham, Dorset), and 250 μl of 20% SDS in a Coplin jar, and then topped up with MilliQ water to 50 ml. The solution was pre-warmed to 70°C in an oven before use. A microarray slide was placed in the slot in the Coplin jar and incubated 70°C for 20 min. The slide was then taken out and placed in a slide rack and immersed in a trough filled with approximately 200 ml of isopropanol to wash. The slide rack was shaken up and down manually for 1 min and then transferred into another trough filled with 200 ml of MilliQ water and again shaken manually for 1 min. The slide was dried using an airbrush. A size 22 X 50 mm Lifter Slip (Erie Scientific Company; Portsmouth, USA) was dipped into first the isopropanol, and then the water and similarly dried. The dry slide was placed facing up in a hybridization chamber (Corning) and the lifter slip was then placed on the array area of the slide and the chamber was sealed with metal clamps before warming up in a 42°C incubator.



Hybridization:

The pre-warmed slide was taken out and 10 µl of 1X genHYB (Genetix; New Milton, Hampshire) solution was added to each reservoir on both ends of the hybridization chamber for the purpose of humidifying the chamber. The pre-warmed hybridization sample was pipetted underneath one edge of the Lifter Slip and let spread throughout the lifter slip area by capillary. The chamber was then re-sealed and incubated at 42°C for 16-18 h.



Washings:

Washing solutions were also pre-warmed at 42°C overnight. These are GenHyb Solution A (1X SSC, 0.2% SDS), GenHyb Solution B (0.2X SSC, 0.2% SDS), and GenHyb Solution C (0.2X SSC).



After overnight incubation, the slide was taken out of the hybridization chamber and dipped into a trough containing 200 ml of pre-warmed GenHyb A solution and the Lifter Slip was slid off gently. The slide was then placed in a rack and this was immersed in pre-warmed GenHyb Solution A, which was then shaken at 42°C at 150 rpm for 5 min. The rack with the slide was then transferred into another trough containing pre-warmed GenHyb Solution B, and similarly shaken at 42°C at 150 rpm for 5 min. The last wash was in pre-warmed GenHyb Solution C shaken again at 42°C at 150 rpm for 30 s. The slide dried using an airbrush.


(Parameters: Chamber type = Corning Microarray Technology- CMT-Hyb chamber, Quantity of label target used = 20, Mass unit = Micro gram, time = 18, Tiny time unit = hours, Volume = 60, Volume unit = Micro litre, temperature = 42) Fused output file with all high quality / non-excluded features included, and any data from replicate probe features combined using the Fusion algorithm.

NOTE - THAT THIS DATA FILE IS NOT 'NORMALIZED' - LEAVING IT STILL SUITABLE FOR CROSS-CHANNEL CORRECTION.

ANY ANALYSIS SHOULD INCLUDE NORMALIZATION WHERE NECESSARY.

THE DATA, IF GENERATED USING DENDRIMER LABELLING IS NORMALLY LINEAR - AND AS SUCH SHOULD NOT NORMALLY BE SUBJECTED TO LOWESS-TYPE TRANSFORMATIONS. Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology SDRF File E-MEXP-1660.sdrf.txt Term Source Name The MGED Ontology ncbitax ArrayExpress The MGED Ontology mo EFO Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ Term Source Version