Investigation Title Transcription profiling of fission yeast cell cycle, cdc25 block and release 1
Comment[Submitted Name] cdc25 block and release 1
Experimental Design cell_cycle_design transcription profiling by array
Experimental Design Term Source REF EFO
Comment[ArrayExpressReleaseDate] 2004-04-01
Comment[AEMIAMESCORE] 4
Comment[ArrayExpressAccession] E-MEXP-58
Comment[MAGETAB TimeStamp_Version] 2011-06-30 21:17:04 Last Changed Rev: 14857
Experimental Factor Name time
Experimental Factor Type time
Experimental Factor Term Source REF
Person Last Name Rustici Lio Mata Burns Penkett Hayles Brazma Nurse Kivinen Bahler
Person First Name Gabriella Pietro Juan Gavin Chris Jacky Alvis Paul Katja Jurg
Person Mid Initials J
Person Email gr2@sanger.ac.uk
Person Phone 01223494861
Person Fax
Person Address Hinxton
Person Affiliation Pombe Functional Genomics Unit EMBL Outstation-Hinxton The Wellcome Trust Sanger Institute The Wellcome Trust Sanger Institute The Wellcome Trust Sanger Institute Cancer Research UK London Research Institute EMBL Outstation-Hinxton Cancer Research UK London Research Institute EMBL Outstation-Hinxton The Wellcome Trust Sanger Institute
Person Roles submitter
Person Roles Term Source REF mo
Quality Control Type
Quality Control Term Source REF
Replicate Type
Replicate Term Source REF
Normalization Type
Normalization Term Source REF
Date of Experiment
Public Release Date 2004-04-01
PubMed ID
Publication DOI
Publication Author List Gabriella Rustici; Juan Mata; Katja Kivinen; Pietro Lio; Chris Penkett; Gavin Burns; Jacky Hayles; Alvis Brazma; Paul Nurse; Jurg Bahler
Publication Title Periodic Gene Expression Program of the Fission Yeast Cell Cycle
Publication Status
Publication Status Term Source REF
Experiment Description Cell cycle timecourse of fission yeast using a whole-culture synchronisation method temperature-sensitive mutant cdc25 nr. 1
Protocol Name P-MEXP-2373 P-MEXP-2315 P-MEXP-2317 P-MEXP-2316 P-MEXP-2318 P-MEXP-3086
Protocol Type nucleic_acid_extraction labeling labeling hybridization bioassay_data_transformation
Protocol Description strain cdc25-22 h- was grown at 25ºC to OD600 ~0.2. The culture was shifted to 36ºC for 3.5 h and quickly shifted back to 25ºC. Samples were taken every 15 min for 5 h. Unsynchronized cells of the same culture before shift to 36ºC were used as reference for all timepoints. 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 -70aC.
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 Ýl of TES (adjust if total cells are >5 ODs), resuspend cells with pipette, immediately add 750 Ýl acidic phenol-chloroform (refrigerated, Sigma P-1944), vortex, and incubate in 65aC heat block (use fume hood!). Then do the next sample in the same way.
4. Incubate all samples in 65aC 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 4aC.
6. Pre-spin 2 ml yellow phase-lock (heavy) tubes (Eppendorf) for 10 sec.
Add 700 Ýl of acidic phenol-chloroform.
7. Take 700 Ýl 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 4aC.
8. Pre-spin 2 ml phase-lock tubes as in step 6.
Add 700 Ýl of chloroform:isoamyl alcohol (24:1)(under fume hood, Sigma C-0549).
9. Take 700 Ýl 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 4aC.
10. Prepare normal 2 ml Eppendorf tubes with 1.5 ml of 100% EtOH (-20aC) and 50 Ýl of 3 M NaAc pH 5.2.
11. Transfer 500 Ýl of water phase from step 9 to the tubes from step 10, vortex 10 sec. Samples can be precipitated at -20aC overnight (or at -70aC for 30 min).
12. Centrifuge for 10 min at 14,000 rpm at RT. Discard SN, add 500 Ýl 70% EtOH (4aC, 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 Ýl of DEPC water, and incubate 1 min at 65aC (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 Ýl to 995 Ýl DEPC water (1:200), set reference with water in 500 Ýl 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 Ýg of RNA in total, but it may be less for RNA isolated under some conditions. Use 100 Ýg 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 -70aC as a backup.
16. Purify 100 Ýg 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 Ýl RNase-free water. Keep on ice!
17. Run 2 Ýl 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 Ýl to 100 Ýl DEPC water (1:50), set reference with water in 50 Ýl 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 Ýg RNA/13.9 Ýl.
20. From each sample, use ~50% of your RNA to make up a reference pool by combining equal amounts (e.g. 40 Ýl) from every timepoint. Mix and make up 12.9 Ýl aliquots stored at -70aC (ready to use for labeling).
Make up 13.9 Ýl aliquots if not using bacterial control RNA for labeling. 21. With the rest of your RNA, make up 12.9 Ýl aliquots of each sample and immediately store at -70aC (ready to use for labeling).Make up 13.9 Ýl 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)
1. Set temperature blocks to 70C and 42C (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 70C 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 42C for 1.5 hrs. Cover with lid for light protection.
10. Add 1.5 l of 1 M NaOH and incubate at 70C 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 100C 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 -20C:
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 -70C 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 (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.
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 Denhardts 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 100C 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.
1. Set temperature blocks to 70C and 42C (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 70C 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 42C for 1.5 hrs. Cover with lid for light protection.
10. Add 1.5 l of 1 M NaOH and incubate at 70C 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 100C 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 -20C:
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 -70C 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 (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.
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 Denhardts 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 100C 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.
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 Ýl of 15 x SSC to each filter. Put microarrays into chamber and incubate at 49aC 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.
Raw data is normalized on an array by array basis using an in-house normalization script.
Reference:
Rachel Lyne, Gavin Burns, Juan Mata, Chris J Penkett, Gabriella Rustici, Dongrong Chen, Cordelia Langford, David Vetrie, Jurg Bahler;
BMC Genomics 2003, 4:27 (10 July 2003).
Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data.
Overview of Script.
-------------------
The script performs three primary tasks:
1) Masking bad spots.
2) Filtering lower quality spots.
3) Applying local normalisation.
1) Masking Bad Spots.
In the GenePix results file, the 'ID' column is used for the gene ID and the 'Name' column is used for information about the type or quality of the PCR spot on the micro-array slide. In the 'Name' column, failed or faulty spots are set to 'A' (absent), and spots with low probe concentration or where the reverse primer is located 2500-3000 bp from the 3' end of the gene are set to 'M' (marginal). Also, in the 'Name' column, 'Q' indicates bacterial controls, 'T' indicates tags or markers, and 'Y' indicates genes that are controls for cross-hybridisation (budding yeast genes of different levels of homology in our case).
This information is then used together with the 'Flags' column in the results file regarding the spot finding algorithm. For example, spots not found are given a flag of '-50', and spots masked as bad by the user are given a flag of '-100' by GenePix. These flags are masked as being absent ('A'). For each spot, a combined flag is produced such that any spot that is absent in either the 'Name' column or the 'Flags' column is given an overall flag of absent ('A'). The combined flag is set to marginal ('M') for any spot that is marginal in the 'Name' column. This option is performed automatically on all data.
2) Filtering lower quality spots.
A background cut-off is applied to discard data from weak signals: spots with < 50% of pixels > 2 SD above median local background signal in one or both channels are flagged absent, unless one channel shows > 95% of the pixels > 2 SD above local background. The SD was calculated using only the lower 55% of the pixel intensities (called SD2 in GenePix Pro), as this measure is less susceptible to being skewed by bright pixels. The default cut-offs of 50% and 95% can be changed in the GUI, and this filtering option can also be switched off.
3) Applying local normalisation.
The script also performs a local normalisation using a sliding square window of spots surrounding each spot. A user-defined minimum number of spots is chosen to be used with which to normalise over (default is 400). The window size default is 16 spots. This means the square contains 33 x 33 spots (1089) surrounding central spots, 33 x 17 spots (561) surrounding spots at the edge of the array, and 17 x 17 spots (289) surrounding spots in a corner of the array. Only spots that are flagged present are used for the normalisation. Hence, using a window of 16 means that sometimes, especially for spots close to the corners of the array, less than 400 spots may actually be used for the normalisation. In cases where the block size chosen is small, the window size is increased up to a user-defined maximum window size (default is 24) so that at least 600 total spots are in the square. This means the block size used with this window change is larger than may be necessary to optimise the chances of having 400 present spots to use for normalisation. This is a heuristic to make the algorithm faster for the majority of spots, since counting the number of present spots in the initial square uses a rel2atively large amount of computational time. If, during normalisation, the number of spots is still found to be less than 400, the window is increased further until the maximum window size is reached. In these cases, the spots that do use less than 400 spots for normalisation are reported in the output log file. The script then calculates a normalisation factor such that the median signal ratio of all measurable spots within the square equals 1, and this factor is then used to scale the signal ratio for the central spot. The default window sizes (minimum: 16 and maximum: 24) and normalisation block sizes (minimum: 400 and initial estimate: 600) can be changed in the GUI.
Protocol Parameters media;max temperature;start Time;min temperature;stop Time; Extracted product;Amplification; Amplification;Label used;Amount of nucleic acid labeled; Amplification;Amount of nucleic acid labeled;Label used; Volume;Temperature;Time;Quantity of label target used;Chamber type;
Protocol Hardware
Protocol Software Initial microarray data processing (data filtering, local normalisation and quality control)
Protocol Contact
Protocol Term Source REF
SDRF File E-MEXP-58.sdrf.txt
Term Source Name ArrayExpress EFO mo
Term Source File http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php
Term Source Version