7 protocols
AccessionNameType
P-MEXP-2317
labeling
1. Set temperature blocks to 70C and 42C (or waterbath). Place all required reagents and samples on ice (experimental and reference RNA aliquots, bacterial RNA ‘cocktail’, LabellingMix*, oligo(dT) primer, and DEPC water, not Superscript enzyme or Cy dyes).
2. Add 1 l of bacterial RNA ‘cocktail’ (in water) and 2.5 l of anchored oligo-dT17 primer (2 μg/μl) to each 12.9 l RNA sample (or 1.25 μl each of random nonamer (dN9) and oligo(dT) primer). Prepare a separate tube with the corresponding reference pool RNA for each timepoint. (For polyA RNA use less water and additional random hexamers).
3. Incubate the reaction mixtures at 70C for 10 min. Place Cy dyes on ice for step 8.
4. Snap-chill the tubes on ice. Spin down reaction mixtures for 15 sec and place the tubes on ice.
5. Make sure the LabellingMix* is completely thawed, then thoroughly vortex, and add 9.6 l of this mix to each tube with RNA (change tips between tubes).
6. With Gilson P2 pipette add 2 l of dCTP Cy3 or Cy5-labelled nucleotide to each tube. Remember which dye you use for experiment vs reference and stick to it for the timecourse. Change tips between tubes. Keep exposure to light of Cy dyes to minimum.
7. With Gilson P2 pipette add 2 l of Superscript II reverse transcriptase (Invitrogen) 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 reaction mixture should now be 30 l. Vortex reactions and spin down.
9. Incubate the reactions at 42C for 1.5 hrs. Cover with lid for light protection.
10. Add 1.5 l of 1 M NaOH and incubate at 70C for 15 min to hydrolyze the RNA (in meantime prepare columns as described in 12.).
11. Add 1.5 l of 1 M HCl and mix to neutralize. Set temperature block to 100C for step 23.
12. Prepare AutoSeq G-50 columns (Amersham), 1 column 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. Note: If using a flip-cap tube, 10-20 l of fluid will remain in the tip of the column after spinning. Blot this fluid from the column using a clean paper towel before applying sample into the column.
- 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.
______________________________________________________________________________
* LabellingMix: Prepare for 20 labelling reactions, store at -20C:
• 120 l 5x first strand buffer (Invitrogen)
• 60 l 0.1M DTT (Invitrogen)
• 12 l dNTP mix (25 mM dATP, dTTP, dGTP, 10 mM dCTP)

13. 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.
14. Pool purified experimental cDNAs with corresponding reference cDNAs (for timecourse experiments first pool and mix all reference samples, then add an equal aliquot of reference to each experimental sample).
15. Add 1/10th volume of 3M NaAc pH 5.2 (6 l) and 3 volumes of RT 100% EtOH (180 l), mix, and precipitate at RT or -70C for 30 min (RT minimizes nucleotide precipitation).
16. Centrifuge at RT for 15 min at 14,000 rpm. Pellet should appear purple. Discard SN, add 100 l of 70% EtOH (4C), 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.
17. Air dry for 5 min at RT. Make a master mix for all your reactions (calculate some extra, e.g. for ½ reaction): ~45 l/reaction of hybridization buffer (5 x SSC, 6 x Denhardt’s 60 mM TrisHCl pH 7.6, 0.12% sarkosyl, 48% formamide; filter sterilized) and 3 l/reaction of polyA DNA (2 g/l, Sigma P0887). Add to each ss cDNA pellet and resuspend pellet with pipette.
18. 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.
P-MEXP-2316
labeling
1. Set temperature blocks to 70C and 42C (or waterbath). Place all required reagents and samples on ice (experimental and reference RNA aliquots, bacterial RNA ‘cocktail’, LabellingMix*, oligo(dT) primer, and DEPC water, not Superscript enzyme or Cy dyes).
2. Add 1 l of bacterial RNA ‘cocktail’ (in water) and 2.5 l of anchored oligo-dT17 primer (2 μg/μl) to each 12.9 l RNA sample (or 1.25 μl each of random nonamer (dN9) and oligo(dT) primer). Prepare a separate tube with the corresponding reference pool RNA for each timepoint. (For polyA RNA use less water and additional random hexamers).
3. Incubate the reaction mixtures at 70C for 10 min. Place Cy dyes on ice for step 8.
4. Snap-chill the tubes on ice. Spin down reaction mixtures for 15 sec and place the tubes on ice.
5. Make sure the LabellingMix* is completely thawed, then thoroughly vortex, and add 9.6 l of this mix to each tube with RNA (change tips between tubes).
6. With Gilson P2 pipette add 2 l of dCTP Cy3 or Cy5-labelled nucleotide to each tube. Remember which dye you use for experiment vs reference and stick to it for the timecourse. Change tips between tubes. Keep exposure to light of Cy dyes to minimum.
7. With Gilson P2 pipette add 2 l of Superscript II reverse transcriptase (Invitrogen) 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 reaction mixture should now be 30 l. Vortex reactions and spin down.
9. Incubate the reactions at 42C for 1.5 hrs. Cover with lid for light protection.
10. Add 1.5 l of 1 M NaOH and incubate at 70C for 15 min to hydrolyze the RNA (in meantime prepare columns as described in 12.).
11. Add 1.5 l of 1 M HCl and mix to neutralize. Set temperature block to 100C for step 23.
12. Prepare AutoSeq G-50 columns (Amersham), 1 column 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. Note: If using a flip-cap tube, 10-20 l of fluid will remain in the tip of the column after spinning. Blot this fluid from the column using a clean paper towel before applying sample into the column.
- 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.
______________________________________________________________________________
* LabellingMix: Prepare for 20 labelling reactions, store at -20C:
• 120 l 5x first strand buffer (Invitrogen)
• 60 l 0.1M DTT (Invitrogen)
• 12 l dNTP mix (25 mM dATP, dTTP, dGTP, 10 mM dCTP)

13. 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.
14. Pool purified experimental cDNAs with corresponding reference cDNAs (for timecourse experiments first pool and mix all reference samples, then add an equal aliquot of reference to each experimental sample).
15. Add 1/10th volume of 3M NaAc pH 5.2 (6 l) and 3 volumes of RT 100% EtOH (180 l), mix, and precipitate at RT or -70C for 30 min (RT minimizes nucleotide precipitation).
16. Centrifuge at RT for 15 min at 14,000 rpm. Pellet should appear purple. Discard SN, add 100 l of 70% EtOH (4C), 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.
17. Air dry for 5 min at RT. Make a master mix for all your reactions (calculate some extra, e.g. for ½ reaction): ~45 l/reaction of hybridization buffer (5 x SSC, 6 x Denhardt’s 60 mM TrisHCl pH 7.6, 0.12% sarkosyl, 48% formamide; filter sterilized) and 3 l/reaction of polyA DNA (2 g/l, Sigma P0887). Add to each ss cDNA pellet and resuspend pellet with pipette.
18. 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.
P-MEXP-2319
image_acquisition
ArrayExpress
P-MEXP-3086
bioassay_data_transformation
ArrayExpress
P-MEXP-2315
nucleic_acid_extraction
1. Harvest cells (usually 25 ml of OD600 ~0.2, adjust volume according to OD).
Centrifuge 2 min at 2000 rpm and discard SN. Snap freeze pellet (liquid nitrogen or dry ice/ethanol). Alternatively, filter cells and snap freeze filter disc. Store cells at -70„aC.
2. Thaw cells on ice (~5 min). Add 1 ml of pre-chilled DEPC water, resuspend cells, and transfer to 2 ml Eppendorf tubes. Spin 10 sec at 5000 rpm and remove SN.
3. To pellet add 750 ƒÝl of TES (adjust if total cells are >5 ODs), resuspend cells with pipette, immediately add 750 ƒÝl acidic phenol-chloroform (refrigerated, Sigma P-1944), vortex, and incubate in 65„aC heat block (use fume hood!). Then do the next sample in the same way.
4. Incubate all samples in 65„aC heat block for 1 hr, vortex 10 sec every 10 min.
5. Place samples on ice for 1 min, vortex 20 sec, and centrifuge for 15 min at 14,000 rpm at 4„aC.
6. Pre-spin 2 ml yellow phase-lock (heavy) tubes (Eppendorf) for 10 sec.
Add 700 ƒÝl of acidic phenol-chloroform.
7. Take 700 ƒÝl of the water phase from step 5 and add to the phase-lock tubes from step 6, thoroughly mix by inverting (no vortexing), and centrifuge 5 min at 14,000 rpm at 4„aC.
8. Pre-spin 2 ml phase-lock tubes as in step 6.
Add 700 ƒÝl of chloroform:isoamyl alcohol (24:1)(under fume hood, Sigma C-0549).
9. Take 700 ƒÝl of the water phase from step 7 and add to the phase-lock tubes from step 8, thoroughly mix by inverting (no vortexing), and centrifuge 5 min at 14,000 rpm at 4„aC.
10. Prepare normal 2 ml Eppendorf tubes with 1.5 ml of 100% EtOH (-20„aC) and 50 ƒÝl of 3 M NaAc pH 5.2.
11. Transfer 500 ƒÝl of water phase from step 9 to the tubes from step 10, vortex 10 sec. Samples can be precipitated at -20„aC overnight (or at -70„aC for 30 min).
12. Centrifuge for 10 min at 14,000 rpm at RT. Discard SN, add 500 ƒÝl 70% EtOH (4„aC, made with DEPC water), don¡¦t vortex, just add, and spin for 1 min (same tube orientation!). Aspirate most SN, spin 5 sec, and remove rest of liquid with pipette. Air dry 5 min at RT.
13. Add 100 ƒÝl of DEPC water, and incubate 1 min at 65„aC (or 10 min at RT). Dissolve pellet first by pipetting up and down (~30x) until no particles are left, then gently vortex 10 sec.
14. Measure OD260/280: add 5 ƒÝl to 995 ƒÝl DEPC water (1:200), set reference with water in 500 ƒÝl glass cell, then measure RNA (OD should be >0.1). Rinse cell with 0.1M NaOH, 0.1M HCl, and thoroughly with ddH20.
15. Expect ~400 ƒÝg of RNA in total, but it may be less for RNA isolated under some conditions. Use 100 ƒÝg of your RNA for Qiagen purification (see step 16). Measure the volume of the remaining RNA, add 3 volumes of 100% EtOH and store at -70„aC as a backup.
16. Purify 100 ƒÝg of each of your RNAs using RNeasy mini spin columns (Qiagen) as described in the RNeasy Mini Handbook (p. 48-49). Elute twice with 30 ƒÝl RNase-free water. Keep on ice!
17. Run 2 ƒÝl of purified RNA on a 1% agarose gel (wipe gel apparatus/tray with RNAse-Zap, rinse with water and use new TBE buffer, use RNAse-free loading buffer made with DEPC water). You should see the two ribosomal bands clean, distinct and without smears.
18. Measure OD260/280 of purified RNA: add 2 ƒÝl to 100 ƒÝl DEPC water (1:50), set reference with water in 50 ƒÝl glass cell, then measure RNA (OD should be >0.1; ratios 260/280 >1.8).
Rinse cell with 0.1M NaOH, 0.1M HCl, and thoroughly with ddH20.
19. Add DEPC water to every sample such that the end concentration is 20 ƒÝg RNA/13.9 ƒÝl.
20. From each sample, use ~50% of your RNA to make up a reference pool by combining equal amounts (e.g. 40 ƒÝl) from every timepoint. Mix and make up 12.9 ƒÝl aliquots stored at -70„aC (ready to use for labeling).
Make up 13.9 ƒÝl aliquots if not using bacterial control RNA for labeling. 21. With the rest of your RNA, make up 12.9 ƒÝl aliquots of each sample and immediately store at -70„aC (ready to use for labeling).Make up 13.9 ƒÝl aliquots if not using bacterial control RNA for labeling.


TES: 10mM Tris pH 7.5; 10mM EDTA pH 8; 0.5% SDS (do not treat Tris stock with DEPC, just use DEPC treated water to make solution; store at RT)
P-MEXP-2318
hybridization
19. 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.
20. Prepare Boekel humid chamber with 8 round Whatman GF/D 25 mm filters, and add 300 ƒÝl of 15 x SSC to each filter. Put microarrays into chamber and incubate at 49„aC for ~16 hrs in Grant Boekel hybridization oven (or alternative hybridization chamber).
21. Remove microarrays from chamber and immediately place into a staining jar filled with wash solution 1 (2 x SSC, filter sterilized, HPLC water). Let coverslip fall off by itself (~15 sec).
22. Put microarrays in slide rack. Wash in a staining jar with ~350 ml of solution 1 at RT for 5 min with gentle shaking.
23. Transfer microarrays in slide rack to wash solution 2 (0.05 x SSC, 0.1% SDS, filter sterilized, HPLC water). Wash at RT for 15 min with gentle shaking as before.
24. Repeat step 23.
25. Transfer microarrays in rack to wash solution 3 (0.05 x SSC, filter sterilized, HPLC water).
Wash at RT for 5 min with gentle shaking as before.
26. Exchange wash solution 3 once to get rid of all SDS.
27. Quickly transfer microarrays in slide rack to a centrifuge and spin at 1000 rpm for 1 min to dry the slides.
28. Store microarrays in light protected slide box at RT. Scan as soon as possible.
P-MEXP-2395
grow
For centrifugal elutriations, 5L of cdc25-22 h- cells were grown at 25ºC to OD600 ~0.2, before loading into a Beckman J6 centrifuge with JE-5.0 elutriation rotor and eluted at 25ºC with 80% fresh EMM, 20% conditioned EMM, 0.005% YE. The eluted fraction was then shifted to 36ºC for 2.5 h and samples were collected every 15 min for ~7 h during the block and after the release. Unsynchronized cells of the same culture before elutriation were used as reference for all timepoints.