Result

Search results for the GEO ID: GSE12415

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Title

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Description

Characteristics

GSM311591

GPL1261

Wild type1

Postnatal Day 0.5 lens tissue, wildtype

Background Srain: 129S3 Gender: Male Tissue: lens Age: 0.5 Days Postnatal

Wild type

Sample_geo_accession	GSM311591
Sample_status	Public on Aug 11 2009
Sample_submission_date	Aug 12 2008
Sample_last_update_date	Aug 18 2008
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Affymatix
Sample_treatment_protocol_ch1	Lens were dissected from day 0.5 animals and placed in RNAlater (Ambion) for 24 hours at 4 degrees Celsius. After 24 hours, the RNAlater was removed and the lens were frozen on dry ice and placed in a -80 degree freezer.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	RNA was isolated using Qiagen RNeasy mini kits. On column DNase digestion was used. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Affymatrix Bioanalyzer system.
Sample_label_ch1	Biotin
Sample_label_protocol_ch1 = Test RNA sample on NanoDrop. Concentration must be 500_g/µL or more. Calculate the volume of RNA sample necessary to start protocol with 5µg. i.e. (5 µg) / (NanoDrop concentration reading in µg/µL)	µL
Sample_label_protocol_ch1	RNA x uL
Sample_label_protocol_ch1	DEPC H20 10-x uL
Sample_label_protocol_ch1	oligo T7 2.0 µL
Sample_label_protocol_ch1	1. Add all components together in a 0.2µL strip tube.
Sample_label_protocol_ch1	2. Mix well and spin down.
Sample_label_protocol_ch1	3. Place in thermalcycler at 70°C for 10 minutes. Should be warming up to 70°C while you prep. the samples.
Sample_label_protocol_ch1	4. Allow thermalcycler to ramp down to 4°C.
Sample_label_protocol_ch1	5. 4°C for 2 minutes. Spin down and put on ice.
Sample_label_protocol_ch1	6. Prepare the 1st Strand MasterMix.
Sample_label_protocol_ch1	Reverse Transcription (RT) 1st Strand
Sample_label_protocol_ch1	You should have thawed and vortexed the components during the 70°C-10 minute step.
Sample_label_protocol_ch1	5X 1st Strand Mix (4.0 µL) /sample
Sample_label_protocol_ch1	0.1M DTT (2.0 µL) /sample
Sample_label_protocol_ch1	10mM dNTPs (1.0 µL) /sample
Sample_label_protocol_ch1	7.0 µL total
Sample_label_protocol_ch1	1. Add 7.0 µL 1st Strand MasterMix to each sample. Mix.
Sample_label_protocol_ch1	2. Put on thermalcycler 42°C-1 Hour. Set timer for 2 minutes.
Sample_label_protocol_ch1	(This setting is actually programmed for 42°C for 1 hour and 3 minutes, to compensate for the extra 2
Sample_label_protocol_ch1	minutes at the beginning before adding the SuperScript II.
Sample_label_protocol_ch1	3. After 2 minutes at 42°C add 1.0 µL of SSII enzyme. (Addition should be done on thermalcycler)
Sample_label_protocol_ch1	4. The rest of the program will run, 1 hour at 42°C.
Sample_label_protocol_ch1	5. Proceed to 2nd Strand.
Sample_label_protocol_ch1	(components should be thawed ~10 minutes before thermalcycler finishes.)
Sample_label_protocol_ch1	Reverse Transcription (RT) 2nd Strand
Sample_label_protocol_ch1	DEPC H20 91.0 µL /sample
Sample_label_protocol_ch1	5X 2nd Strand Mix 30.0 µL /sample
Sample_label_protocol_ch1	E. coli DNA Polymerase I (DNA Pol I) 4.0 µL /sample
Sample_label_protocol_ch1	10mM dNTPs 3.0 µL /sample
Sample_label_protocol_ch1	E. coli DNA ligase (ligase) 1.0 µL /sample
Sample_label_protocol_ch1	RNase H 1.0 µL /sample
Sample_label_protocol_ch1	1. Put samples on thermalcycler, 16°C for 2 hours.
Sample_label_protocol_ch1	2. At 2 hours, add 2.0 µL of T4 DNA Polymerase. Mix.
Sample_label_protocol_ch1	3. Run additional 5 minutes at 16°C.
Sample_label_protocol_ch1	4. Transfer samples to 1.5mL tube with 10.0 µL of 0.5M EDTA. Vortex.
Sample_label_protocol_ch1	5. Use cDNA Clean-Up Module to clean up samples.
Sample_label_protocol_ch1	Clean-Up of Double Stranded cDNA
Sample_label_protocol_ch1	Necessary components:
Sample_label_protocol_ch1	1.5mL tube with cDNA and EDTA, mixed.
Sample_label_protocol_ch1	cDNA Binding Buffer
Sample_label_protocol_ch1	cDNA Wash Buffer (ethanol added by MCF)
Sample_label_protocol_ch1	cDNA Elution Buffer
Sample_label_protocol_ch1	cDNA clean-up column (in 4°C Deli Fridge)
Sample_label_protocol_ch1	Empty Collection Tube (provided in clean-up module)
Sample_label_protocol_ch1	Empty 1.5mL tube labeled with sample name for collecting final eluate.
Sample_label_protocol_ch1	MicroFuge 18 Centrifuge to spin down samples.
Sample_label_protocol_ch1	cDNA Wash Buffer is supplied as a concentrate and you have to add ethanol before using. Check the tube before use to make sure that etOH has been added.
Sample_label_protocol_ch1	1. Label Collection Tube with sample name (on side).
Sample_label_protocol_ch1	2. Add 600.0 µL of cDNA Binding Buffer (yellow) into tube with sample and EDTA.
Sample_label_protocol_ch1	3. Vortex. Spin down briefly (2 sec.)
Sample_label_protocol_ch1	4. Add 500.0 µL sample-Binding Buffer mix onto column.
Sample_label_protocol_ch1	5. Spin in MicroFuge 18 Centrifuge, 14,000rpm for 1 minute.
Sample_label_protocol_ch1	6. Pour eluate down sink. Keep same collection tube.
Sample_label_protocol_ch1	7. Add the remaining cDNA-Binding Buffer mix onto column.
Sample_label_protocol_ch1	8. Spin 14K rpm for 1 minute.
Sample_label_protocol_ch1	9. Discard that collection tube and place column into new, labeled tube.
Sample_label_protocol_ch1	10. Add 720.0 µL cDNA Wash Buffer (ETOH added) to the column.
Sample_label_protocol_ch1	11. Spin 14K rpm for 1 minute.
Sample_label_protocol_ch1	12. Discard eluate into sink. Replace column into collection tube.
Sample_label_protocol_ch1	13. Spin empty column with the lid open for 5 minutes at 14K rpm.
Sample_label_protocol_ch1	14. Place column into labeled 1.5mL tube. Discard collection tube.
Sample_label_protocol_ch1	15. Air dry column for an additional minute (cover with KimWipe).
Sample_label_protocol_ch1	16. Add 14.0 µL Elution Buffer onto the center of the column.
Sample_label_protocol_ch1	17. Let sit at room temp for 1 minute.
Sample_label_protocol_ch1	18. Spin at 14K for 1 minute.
Sample_label_protocol_ch1	19. Discard column.
Sample_label_protocol_ch1	20. Measure the eluate volume.
Sample_label_protocol_ch1	21. Bring volume up to 20.0 µL with DEPC H20.
Sample_label_protocol_ch1	22. Store at -20ºC until ready to proceed with IVT step.
Sample_label_protocol_ch1	InVitro Transcription Reaction (IVT): production of biotin-labeled cRNA
Sample_label_protocol_ch1	cDNA should be at 20.0 µL before adding IVT MasterMix components.
Sample_label_protocol_ch1	IVT kit components should be thawed to room temp. ~20 minutes before using.
Sample_label_protocol_ch1	10X IVT Labeling Buffer (may have precipitate, mix well.) (4.0 µL) /sample
Sample_label_protocol_ch1	IVT Labeling NTP Mix (12.0 µL) /sample
Sample_label_protocol_ch1	IVT Labeling Enzyme Mix (4.0 µL) /sample
Sample_label_protocol_ch1	1. Mix components. Spin briefly.
Sample_label_protocol_ch1	2. Add 20.0 µL IVT MasterMix to each sample. Mix by pipetting.
Sample_label_protocol_ch1	3. 37 ºC for 16 hours in thermalcycler.
Sample_label_protocol_ch1	4. Clean-up cRNA with cRNA Clean-Up Module.
Sample_label_protocol_ch1	Clean-Up of Biotinylated cRNA with cRNA Clean-Up Module
Sample_label_protocol_ch1	Necessary Components:
Sample_label_protocol_ch1	cRNA Binding Buffer
Sample_label_protocol_ch1	cRNA Wash Buffer (ethanol added by MCF)
Sample_label_protocol_ch1	cRNA clean-up column (stored at 4°C in deli fridge)
Sample_label_protocol_ch1	Collection Tube (labeled with sample name on side)
Sample_label_protocol_ch1	DEPC H20
Sample_label_protocol_ch1	100% Ethanol
Sample_label_protocol_ch1	80% Ethanol
Sample_label_protocol_ch1	2 clean, labeled 1.5mL tubes
Sample_label_protocol_ch1	1. Transfer cRNA samples into clean, labeled 1.5mL tubes for clean-up.
Sample_label_protocol_ch1	2. Add 60.0 µL to each sample. Vortex to mix and spin down.
Sample_label_protocol_ch1	3. Add 350.0 µL cRNA Binding Buffer to each sample. Vortex and spin down.
Sample_label_protocol_ch1	4. Add 250.0 µL 100% ETOH to each sample. Mix by pipetting.
Sample_label_protocol_ch1	5. Add cRNA-Binding Buffer-ETOH mix onto column (it all fits).
Sample_label_protocol_ch1	6. Spin at 14,000rpm on “Short Spin” setting (30 seconds).
Sample_label_protocol_ch1	7. Place columns into clean, labeled collection tube. Discard previous tubes with eluate.
Sample_label_protocol_ch1	8. Add 500.0 µL cRNA Wash Buffer to each column.
Sample_label_protocol_ch1	9. Spin at 14K for “Short Spin”.
Sample_label_protocol_ch1	10. Discard eluate in sink but replace column into collection tube.
Sample_label_protocol_ch1	11. Add 500.0 µL 80% ETOH onto column.
Sample_label_protocol_ch1	12. “Short Spin” at 14K.
Sample_label_protocol_ch1	13. Discard eluate and replace columns into collection tube again.
Sample_label_protocol_ch1	14. Spin 14K for 5 minutes to dry column.
Sample_label_protocol_ch1	15. Put column into clean, labeled 1.5mL tube. (This tube should have the request number, the term “IVT” and the sample name on top. On the side put the PI last name and the date.)
Sample_label_protocol_ch1	16. Pipet 11.0 µL DEPC H20 onto center of column.
Sample_label_protocol_ch1	17. Spin 14K for 1 minute.
Sample_label_protocol_ch1	18. Add another 10.0 µL DEPC H20 onto center of column.
Sample_label_protocol_ch1	19. Spin 14K for 1 minute.
Sample_label_protocol_ch1	20. Measure volume of eluate. Record the volume in your lab notebook.
Sample_label_protocol_ch1	21. NanoDrop samples. Dilute with DEPC H20 if you need to, to get concentration into read-able range for NanoDrop (under 3000_g).
Sample_label_protocol_ch1	22. Make a dilution of the IVT samples for testing on the Agilent.
Sample_label_protocol_ch1	Perform calculations to determine adjusted cRNA amount. Enter the total µg into MAID. Perform calculations for Fragmentation step and record the volumes in your lab notebook. Calculations are done with the IVT NanoDrop adjusted cRNA concentrations acquired with the formula provided by Affy.
Sample_label_protocol_ch1	Fragmentation:
Sample_label_protocol_ch1	cRNA (X uL) 15ug
Sample_label_protocol_ch1	DEPC H20(variable) (32-x) µL
Sample_label_protocol_ch1	5X Fragmentation Buffer 8.0 µL
Sample_label_protocol_ch1	40.0 uL
Sample_label_protocol_ch1	1. Add 8.0 µL 5X Frag. Buffer to each empty 1.5mL tube.
Sample_label_protocol_ch1	2. Add the required amount of water to each tube. This amount will depend on the concentration of cRNA- how much cRNA to make 15 µg of fragmented cRNA, then how much DEPC H20 to bring the final volume to 40.0 µL?
Sample_label_protocol_ch1	3. Add the required amount of cRNA to each tube.
Sample_label_protocol_ch1	4. Make sure the 1.5mL tube is clearly labeled FRAG so that you can tell it apart from the IVT cRNA tube.
Sample_label_protocol_ch1	5. Put the FRAG tube onto the 99°C heat block for 35 minutes.
Sample_label_protocol_ch1	6. After fragmentation put the tube on ice. Let it cool about 5 minutes on ice.
Sample_label_protocol_ch1	7. Spin in the MicroFuge at max. speed to get the condensation out of the lid.
Sample_label_protocol_ch1	8. Vortex and spin.
Sample_label_protocol_ch1	9. Test FRAG on NanoDrop. Needs to be 15 µg or more. (15 µg/40 µL=0.375 µg/µL or more)
Sample_label_protocol_ch1	Put the FRAG into hybridization cocktail and store at -20°C until you are ready to load it onto a chip.
Sample_hyb_protocol	Euk 20X must be heated 5 minutes before using it. Vortex before using and spin down. Put on ice when you’re finished with it but not before adding it to MasterMix.
Sample_hyb_protocol	(multiply all by 3.5) MasterMix components
Sample_hyb_protocol	2X Hybe Buffer 150.0 µL 525.0 µL
Sample_hyb_protocol	DEPC H20 54.0 µL (to bring volume up to 300.0µL) 189.0 µL
Sample_hyb_protocol	100% DMSO 30.0 µL 105.0 µL
Sample_hyb_protocol	Eukaryotic 20X Control *** 15.0 µL 52.5 µL
Sample_hyb_protocol	Oligo B2 5.0 µL 17.5 µL
Sample_hyb_protocol	Herring Sperm DNA 3.0 µL 10.5 µL
Sample_hyb_protocol	BSA 3.0 µL 10.5 µL
Sample_hyb_protocol	cRNAfragmented 40.0 µL (if you retrieve all from NanoDrop) you have these separate
Sample_hyb_protocol	1. Keep cRNA in tubes labeled FRAG.
Sample_hyb_protocol	2. Prepare MasterMix in 15mL corning tube.
Sample_hyb_protocol	3. Mix after each component by pipetting several times.
Sample_hyb_protocol	4. Add 260.0 µL of the Hybe MasterMix to each fragmented sample. You total volume should be 300.0 µL.
Sample_hyb_protocol	5. If you are not hybridizing right away, store the cRNA-HybeCocktail mix in the -20°C until you’re ready to use it.
Sample_hyb_protocol	HYBRIDIZATION
Sample_hyb_protocol	Thaw Hybe Cocktails if they are frozen before heating. Pull arrays out of 4°C Deli Fridge 30 minutes before you want to load them. They must be at room temperature before you pierce the septa to prevent leakage.
Sample_hyb_protocol	1. Put tube hats on to prevent popping open at high heat.
Sample_hyb_protocol	2. Place the tubes into the 95°C heating block for 5 minutes.
Sample_hyb_protocol	3. Move the tubes to the 45°C heating block, again for 5 minutes.
Sample_hyb_protocol	4. Spin in MicroFuge at max. speed for 5 minutes.
Sample_hyb_protocol	5. Load 250.0 µL into each array.
Sample_hyb_protocol	6. Put in Hybe Oven, set at 60rpm and 45°C for 16 hours.
Sample_hyb_protocol	After 16 hours of hybridization, remove the hybridization cocktail from the probe array and fill the probe array completely with the appropriate volume of Non-Stringent Wash Buffer (Wash Buffer A)
Sample_hyb_protocol	Wash Buffer A: Non-Stringent Wash Buffer
Sample_hyb_protocol	(6X SSPE, 0.01% Tween-20)
Sample_hyb_protocol	For 1,000 mL:
Sample_hyb_protocol	300 mL of 20X SSPE
Sample_hyb_protocol	1.0 mL of 10% Tween-20
Sample_hyb_protocol	699 mL of water
Sample_hyb_protocol	Filter through a 0.2 μm filter
Sample_hyb_protocol	EukGE-WS2v5*
Sample_hyb_protocol	Midi_euk2*
Sample_hyb_protocol	10 cycles of 2 mixes/cycle with Wash
Sample_hyb_protocol	Buffer A at 30°C 6 cycles of 15 mixes/cycle with
Sample_hyb_protocol	Wash Buffer B at 50°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in SAPE solution at 35°C
Sample_hyb_protocol	10 cycles of 4 mixes/cycle with Wash Buffer A at 30°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in antibody solution at 35°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in SAPE solution at 35°C
Sample_hyb_protocol	15 cycles of 4 mixes/cycle with Wash Buffer A at 35°C. The holding temperature is 25°C
Sample_scan_protocol	[Scanner]
Sample_scan_protocol	Pixel Size 1.56
Sample_scan_protocol	hardware: GeneChip Scanner 3000
Sample_scan_protocol	Affymetrix® Microarray Suite
Sample_scan_protocol	Filter 570
Sample_scan_protocol	Scanner ID 50101490
Sample_scan_protocol	Number of Scans 1
Sample_scan_protocol	Scanner Type M10
Sample_scan_protocol	As affymetrix protocol.
Sample_data_processing	Data in value is raw data (Mas5 scaled) unaltered by normalizations.
Sample_platform_id	GPL1261
Sample_contact_name	Xiangyin,,kong
Sample_contact_email	xykong@sibs.ac.cn
Sample_contact_laboratory	molecular genetics
Sample_contact_department	molecular genetics
Sample_contact_institute	Institute of health science
Sample_contact_address	No.225 yueyang road
Sample_contact_city	Shanghai
Sample_contact_zip/postal_code	200031
Sample_contact_country	China
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM311nnn/GSM311591/suppl/GSM311591.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM311nnn/GSM311591/suppl/GSM311591.CHP.gz
Sample_series_id	GSE12415
Sample_data_row_count	45101

GSM311592

GPL1261

HSF4 knockout1

Postnatal Day 0.5 lens tissue, HSF4 knockout

Background Srain: 129S3 Gender: Male Tissue: lens Age: 0.5 Days Postnatal

HSF4 knockout

Sample_geo_accession	GSM311592
Sample_status	Public on Aug 11 2009
Sample_submission_date	Aug 12 2008
Sample_last_update_date	Aug 18 2008
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_biomaterial_provider_ch1	Affymatix
Sample_treatment_protocol_ch1	Lens were dissected from day 0.5 animals and placed in RNAlater (Ambion) for 24 hours at 4 degrees Celsius. After 24 hours, the RNAlater was removed and the lens were frozen on dry ice and placed in a -80 degree freezer.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	RNA was isolated using Qiagen RNeasy mini kits. On column DNase digestion was used. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Affymatrix Bioanalyzer system.
Sample_label_ch1	Biotin
Sample_label_protocol_ch1 = Test RNA sample on NanoDrop. Concentration must be 500_g/µL or more. Calculate the volume of RNA sample necessary to start protocol with 5µg. i.e. (5 µg) / (NanoDrop concentration reading in µg/µL)	µL
Sample_label_protocol_ch1	RNA x uL
Sample_label_protocol_ch1	DEPC H20 10-x uL
Sample_label_protocol_ch1	oligo T7 2.0 µL
Sample_label_protocol_ch1	1. Add all components together in a 0.2µL strip tube.
Sample_label_protocol_ch1	2. Mix well and spin down.
Sample_label_protocol_ch1	3. Place in thermalcycler at 70°C for 10 minutes. Should be warming up to 70°C while you prep. the samples.
Sample_label_protocol_ch1	4. Allow thermalcycler to ramp down to 4°C.
Sample_label_protocol_ch1	5. 4°C for 2 minutes. Spin down and put on ice.
Sample_label_protocol_ch1	6. Prepare the 1st Strand MasterMix.
Sample_label_protocol_ch1	Reverse Transcription (RT) 1st Strand
Sample_label_protocol_ch1	You should have thawed and vortexed the components during the 70°C-10 minute step.
Sample_label_protocol_ch1	5X 1st Strand Mix (4.0 µL) /sample
Sample_label_protocol_ch1	0.1M DTT (2.0 µL) /sample
Sample_label_protocol_ch1	10mM dNTPs (1.0 µL) /sample
Sample_label_protocol_ch1	7.0 µL total
Sample_label_protocol_ch1	1. Add 7.0 µL 1st Strand MasterMix to each sample. Mix.
Sample_label_protocol_ch1	2. Put on thermalcycler 42°C-1 Hour. Set timer for 2 minutes.
Sample_label_protocol_ch1	(This setting is actually programmed for 42°C for 1 hour and 3 minutes, to compensate for the extra 2
Sample_label_protocol_ch1	minutes at the beginning before adding the SuperScript II.
Sample_label_protocol_ch1	3. After 2 minutes at 42°C add 1.0 µL of SSII enzyme. (Addition should be done on thermalcycler)
Sample_label_protocol_ch1	4. The rest of the program will run, 1 hour at 42°C.
Sample_label_protocol_ch1	5. Proceed to 2nd Strand.
Sample_label_protocol_ch1	(components should be thawed ~10 minutes before thermalcycler finishes.)
Sample_label_protocol_ch1	Reverse Transcription (RT) 2nd Strand
Sample_label_protocol_ch1	DEPC H20 91.0 µL /sample
Sample_label_protocol_ch1	5X 2nd Strand Mix 30.0 µL /sample
Sample_label_protocol_ch1	E. coli DNA Polymerase I (DNA Pol I) 4.0 µL /sample
Sample_label_protocol_ch1	10mM dNTPs 3.0 µL /sample
Sample_label_protocol_ch1	E. coli DNA ligase (ligase) 1.0 µL /sample
Sample_label_protocol_ch1	RNase H 1.0 µL /sample
Sample_label_protocol_ch1	1. Put samples on thermalcycler, 16°C for 2 hours.
Sample_label_protocol_ch1	2. At 2 hours, add 2.0 µL of T4 DNA Polymerase. Mix.
Sample_label_protocol_ch1	3. Run additional 5 minutes at 16°C.
Sample_label_protocol_ch1	4. Transfer samples to 1.5mL tube with 10.0 µL of 0.5M EDTA. Vortex.
Sample_label_protocol_ch1	5. Use cDNA Clean-Up Module to clean up samples.
Sample_label_protocol_ch1	Clean-Up of Double Stranded cDNA
Sample_label_protocol_ch1	Necessary components:
Sample_label_protocol_ch1	1.5mL tube with cDNA and EDTA, mixed.
Sample_label_protocol_ch1	cDNA Binding Buffer
Sample_label_protocol_ch1	cDNA Wash Buffer (ethanol added by MCF)
Sample_label_protocol_ch1	cDNA Elution Buffer
Sample_label_protocol_ch1	cDNA clean-up column (in 4°C Deli Fridge)
Sample_label_protocol_ch1	Empty Collection Tube (provided in clean-up module)
Sample_label_protocol_ch1	Empty 1.5mL tube labeled with sample name for collecting final eluate.
Sample_label_protocol_ch1	MicroFuge 18 Centrifuge to spin down samples.
Sample_label_protocol_ch1	cDNA Wash Buffer is supplied as a concentrate and you have to add ethanol before using. Check the tube before use to make sure that etOH has been added.
Sample_label_protocol_ch1	1. Label Collection Tube with sample name (on side).
Sample_label_protocol_ch1	2. Add 600.0 µL of cDNA Binding Buffer (yellow) into tube with sample and EDTA.
Sample_label_protocol_ch1	3. Vortex. Spin down briefly (2 sec.)
Sample_label_protocol_ch1	4. Add 500.0 µL sample-Binding Buffer mix onto column.
Sample_label_protocol_ch1	5. Spin in MicroFuge 18 Centrifuge, 14,000rpm for 1 minute.
Sample_label_protocol_ch1	6. Pour eluate down sink. Keep same collection tube.
Sample_label_protocol_ch1	7. Add the remaining cDNA-Binding Buffer mix onto column.
Sample_label_protocol_ch1	8. Spin 14K rpm for 1 minute.
Sample_label_protocol_ch1	9. Discard that collection tube and place column into new, labeled tube.
Sample_label_protocol_ch1	10. Add 720.0 µL cDNA Wash Buffer (ETOH added) to the column.
Sample_label_protocol_ch1	11. Spin 14K rpm for 1 minute.
Sample_label_protocol_ch1	12. Discard eluate into sink. Replace column into collection tube.
Sample_label_protocol_ch1	13. Spin empty column with the lid open for 5 minutes at 14K rpm.
Sample_label_protocol_ch1	14. Place column into labeled 1.5mL tube. Discard collection tube.
Sample_label_protocol_ch1	15. Air dry column for an additional minute (cover with KimWipe).
Sample_label_protocol_ch1	16. Add 14.0 µL Elution Buffer onto the center of the column.
Sample_label_protocol_ch1	17. Let sit at room temp for 1 minute.
Sample_label_protocol_ch1	18. Spin at 14K for 1 minute.
Sample_label_protocol_ch1	19. Discard column.
Sample_label_protocol_ch1	20. Measure the eluate volume.
Sample_label_protocol_ch1	21. Bring volume up to 20.0 µL with DEPC H20.
Sample_label_protocol_ch1	22. Store at -20ºC until ready to proceed with IVT step.
Sample_label_protocol_ch1	InVitro Transcription Reaction (IVT): production of biotin-labeled cRNA
Sample_label_protocol_ch1	cDNA should be at 20.0 µL before adding IVT MasterMix components.
Sample_label_protocol_ch1	IVT kit components should be thawed to room temp. ~20 minutes before using.
Sample_label_protocol_ch1	10X IVT Labeling Buffer (may have precipitate, mix well.) (4.0 µL) /sample
Sample_label_protocol_ch1	IVT Labeling NTP Mix (12.0 µL) /sample
Sample_label_protocol_ch1	IVT Labeling Enzyme Mix (4.0 µL) /sample
Sample_label_protocol_ch1	1. Mix components. Spin briefly.
Sample_label_protocol_ch1	2. Add 20.0 µL IVT MasterMix to each sample. Mix by pipetting.
Sample_label_protocol_ch1	3. 37 ºC for 16 hours in thermalcycler.
Sample_label_protocol_ch1	4. Clean-up cRNA with cRNA Clean-Up Module.
Sample_label_protocol_ch1	Clean-Up of Biotinylated cRNA with cRNA Clean-Up Module
Sample_label_protocol_ch1	Necessary Components:
Sample_label_protocol_ch1	cRNA Binding Buffer
Sample_label_protocol_ch1	cRNA Wash Buffer (ethanol added by MCF)
Sample_label_protocol_ch1	cRNA clean-up column (stored at 4°C in deli fridge)
Sample_label_protocol_ch1	Collection Tube (labeled with sample name on side)
Sample_label_protocol_ch1	DEPC H20
Sample_label_protocol_ch1	100% Ethanol
Sample_label_protocol_ch1	80% Ethanol
Sample_label_protocol_ch1	2 clean, labeled 1.5mL tubes
Sample_label_protocol_ch1	1. Transfer cRNA samples into clean, labeled 1.5mL tubes for clean-up.
Sample_label_protocol_ch1	2. Add 60.0 µL to each sample. Vortex to mix and spin down.
Sample_label_protocol_ch1	3. Add 350.0 µL cRNA Binding Buffer to each sample. Vortex and spin down.
Sample_label_protocol_ch1	4. Add 250.0 µL 100% ETOH to each sample. Mix by pipetting.
Sample_label_protocol_ch1	5. Add cRNA-Binding Buffer-ETOH mix onto column (it all fits).
Sample_label_protocol_ch1	6. Spin at 14,000rpm on “Short Spin” setting (30 seconds).
Sample_label_protocol_ch1	7. Place columns into clean, labeled collection tube. Discard previous tubes with eluate.
Sample_label_protocol_ch1	8. Add 500.0 µL cRNA Wash Buffer to each column.
Sample_label_protocol_ch1	9. Spin at 14K for “Short Spin”.
Sample_label_protocol_ch1	10. Discard eluate in sink but replace column into collection tube.
Sample_label_protocol_ch1	11. Add 500.0 µL 80% ETOH onto column.
Sample_label_protocol_ch1	12. “Short Spin” at 14K.
Sample_label_protocol_ch1	13. Discard eluate and replace columns into collection tube again.
Sample_label_protocol_ch1	14. Spin 14K for 5 minutes to dry column.
Sample_label_protocol_ch1	15. Put column into clean, labeled 1.5mL tube. (This tube should have the request number, the term “IVT” and the sample name on top. On the side put the PI last name and the date.)
Sample_label_protocol_ch1	16. Pipet 11.0 µL DEPC H20 onto center of column.
Sample_label_protocol_ch1	17. Spin 14K for 1 minute.
Sample_label_protocol_ch1	18. Add another 10.0 µL DEPC H20 onto center of column.
Sample_label_protocol_ch1	19. Spin 14K for 1 minute.
Sample_label_protocol_ch1	20. Measure volume of eluate. Record the volume in your lab notebook.
Sample_label_protocol_ch1	21. NanoDrop samples. Dilute with DEPC H20 if you need to, to get concentration into read-able range for NanoDrop (under 3000_g).
Sample_label_protocol_ch1	22. Make a dilution of the IVT samples for testing on the Agilent.
Sample_label_protocol_ch1	Perform calculations to determine adjusted cRNA amount. Enter the total µg into MAID. Perform calculations for Fragmentation step and record the volumes in your lab notebook. Calculations are done with the IVT NanoDrop adjusted cRNA concentrations acquired with the formula provided by Affy.
Sample_label_protocol_ch1	Fragmentation:
Sample_label_protocol_ch1	cRNA (X uL) 15ug
Sample_label_protocol_ch1	DEPC H20(variable) (32-x) µL
Sample_label_protocol_ch1	5X Fragmentation Buffer 8.0 µL
Sample_label_protocol_ch1	40.0 uL
Sample_label_protocol_ch1	1. Add 8.0 µL 5X Frag. Buffer to each empty 1.5mL tube.
Sample_label_protocol_ch1	2. Add the required amount of water to each tube. This amount will depend on the concentration of cRNA- how much cRNA to make 15 µg of fragmented cRNA, then how much DEPC H20 to bring the final volume to 40.0 µL?
Sample_label_protocol_ch1	3. Add the required amount of cRNA to each tube.
Sample_label_protocol_ch1	4. Make sure the 1.5mL tube is clearly labeled FRAG so that you can tell it apart from the IVT cRNA tube.
Sample_label_protocol_ch1	5. Put the FRAG tube onto the 99°C heat block for 35 minutes.
Sample_label_protocol_ch1	6. After fragmentation put the tube on ice. Let it cool about 5 minutes on ice.
Sample_label_protocol_ch1	7. Spin in the MicroFuge at max. speed to get the condensation out of the lid.
Sample_label_protocol_ch1	8. Vortex and spin.
Sample_label_protocol_ch1	9. Test FRAG on NanoDrop. Needs to be 15 µg or more. (15 µg/40 µL=0.375 µg/µL or more)
Sample_label_protocol_ch1	Put the FRAG into hybridization cocktail and store at -20°C until you are ready to load it onto a chip.
Sample_hyb_protocol	Euk 20X must be heated 5 minutes before using it. Vortex before using and spin down. Put on ice when you’re finished with it but not before adding it to MasterMix.
Sample_hyb_protocol	(multiply all by 3.5) MasterMix components
Sample_hyb_protocol	2X Hybe Buffer 150.0 µL 525.0 µL
Sample_hyb_protocol	DEPC H20 54.0 µL (to bring volume up to 300.0µL) 189.0 µL
Sample_hyb_protocol	100% DMSO 30.0 µL 105.0 µL
Sample_hyb_protocol	Eukaryotic 20X Control *** 15.0 µL 52.5 µL
Sample_hyb_protocol	Oligo B2 5.0 µL 17.5 µL
Sample_hyb_protocol	Herring Sperm DNA 3.0 µL 10.5 µL
Sample_hyb_protocol	BSA 3.0 µL 10.5 µL
Sample_hyb_protocol	cRNAfragmented 40.0 µL (if you retrieve all from NanoDrop) you have these separate
Sample_hyb_protocol	1. Keep cRNA in tubes labeled FRAG.
Sample_hyb_protocol	2. Prepare MasterMix in 15mL corning tube.
Sample_hyb_protocol	3. Mix after each component by pipetting several times.
Sample_hyb_protocol	4. Add 260.0 µL of the Hybe MasterMix to each fragmented sample. You total volume should be 300.0 µL.
Sample_hyb_protocol	5. If you are not hybridizing right away, store the cRNA-HybeCocktail mix in the -20°C until you’re ready to use it.
Sample_hyb_protocol	HYBRIDIZATION
Sample_hyb_protocol	Thaw Hybe Cocktails if they are frozen before heating. Pull arrays out of 4°C Deli Fridge 30 minutes before you want to load them. They must be at room temperature before you pierce the septa to prevent leakage.
Sample_hyb_protocol	1. Put tube hats on to prevent popping open at high heat.
Sample_hyb_protocol	2. Place the tubes into the 95°C heating block for 5 minutes.
Sample_hyb_protocol	3. Move the tubes to the 45°C heating block, again for 5 minutes.
Sample_hyb_protocol	4. Spin in MicroFuge at max. speed for 5 minutes.
Sample_hyb_protocol	5. Load 250.0 µL into each array.
Sample_hyb_protocol	6. Put in Hybe Oven, set at 60rpm and 45°C for 16 hours.
Sample_hyb_protocol	After 16 hours of hybridization, remove the hybridization cocktail from the probe array and fill the probe array completely with the appropriate volume of Non-Stringent Wash Buffer (Wash Buffer A)
Sample_hyb_protocol	Wash Buffer A: Non-Stringent Wash Buffer
Sample_hyb_protocol	(6X SSPE, 0.01% Tween-20)
Sample_hyb_protocol	For 1,000 mL:
Sample_hyb_protocol	300 mL of 20X SSPE
Sample_hyb_protocol	1.0 mL of 10% Tween-20
Sample_hyb_protocol	699 mL of water
Sample_hyb_protocol	Filter through a 0.2 μm filter
Sample_hyb_protocol	EukGE-WS2v5*
Sample_hyb_protocol	Midi_euk2*
Sample_hyb_protocol	10 cycles of 2 mixes/cycle with Wash
Sample_hyb_protocol	Buffer A at 30°C 6 cycles of 15 mixes/cycle with
Sample_hyb_protocol	Wash Buffer B at 50°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in SAPE solution at 35°C
Sample_hyb_protocol	10 cycles of 4 mixes/cycle with Wash Buffer A at 30°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in antibody solution at 35°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in SAPE solution at 35°C
Sample_hyb_protocol	15 cycles of 4 mixes/cycle with Wash Buffer A at 35°C. The holding temperature is 25°C
Sample_scan_protocol	[Scanner]
Sample_scan_protocol	Pixel Size 1.56
Sample_scan_protocol	hardware: GeneChip Scanner 3000
Sample_scan_protocol	Affymetrix® Microarray Suite
Sample_scan_protocol	Filter 570
Sample_scan_protocol	Scanner ID 50101490
Sample_scan_protocol	Number of Scans 1
Sample_scan_protocol	Scanner Type M10
Sample_scan_protocol	As affymetrix protocol.
Sample_data_processing	Data in value is raw data (Mas5 scaled) unaltered by normalizations.
Sample_platform_id	GPL1261
Sample_contact_name	Xiangyin,,kong
Sample_contact_email	xykong@sibs.ac.cn
Sample_contact_laboratory	molecular genetics
Sample_contact_department	molecular genetics
Sample_contact_institute	Institute of health science
Sample_contact_address	No.225 yueyang road
Sample_contact_city	Shanghai
Sample_contact_zip/postal_code	200031
Sample_contact_country	China
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM311nnn/GSM311592/suppl/GSM311592.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM311nnn/GSM311592/suppl/GSM311592.CHP.gz
Sample_series_id	GSE12415
Sample_data_row_count	45101

GSM312177

GPL1261

Wild type2

Postnatal Day 0.5 lens tissue, wildtype

Background Srain: 129S3 Gender: Male Tissue: lens Age: 0.5 Days Postnatal

Wild type2

Sample_geo_accession	GSM312177
Sample_status	Public on Aug 11 2009
Sample_submission_date	Aug 13 2008
Sample_last_update_date	Aug 18 2008
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_treatment_protocol_ch1	Lens were dissected from day 0.5 animals and placed in RNAlater (Ambion) for 24 hours at 4 degrees Celsius. After 24 hours, the RNAlater was removed and the lens were frozen on dry ice and placed in a -80 degree freezer.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	RNA was isolated using Qiagen RNeasy mini kits. On column DNase digestion was used. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Affymatrix Bioanalyzer system.
Sample_label_ch1	Biotin
Sample_label_protocol_ch1 = Test RNA sample on NanoDrop. Concentration must be 500_g/µL or more. Calculate the volume of RNA sample necessary to start protocol with 5µg. i.e. (5 µg) / (NanoDrop concentration reading in µg/µL)	µL
Sample_label_protocol_ch1	RNA x uL
Sample_label_protocol_ch1	DEPC H20 10-x uL
Sample_label_protocol_ch1	oligo T7 2.0 µL
Sample_label_protocol_ch1	1. Add all components together in a 0.2µL strip tube.
Sample_label_protocol_ch1	2. Mix well and spin down.
Sample_label_protocol_ch1	3. Place in thermalcycler at 70°C for 10 minutes. Should be warming up to 70°C while you prep. the samples.
Sample_label_protocol_ch1	4. Allow thermalcycler to ramp down to 4°C.
Sample_label_protocol_ch1	5. 4°C for 2 minutes. Spin down and put on ice.
Sample_label_protocol_ch1	6. Prepare the 1st Strand MasterMix.
Sample_label_protocol_ch1	Reverse Transcription (RT) 1st Strand
Sample_label_protocol_ch1	You should have thawed and vortexed the components during the 70°C-10 minute step.
Sample_label_protocol_ch1	5X 1st Strand Mix (4.0 µL) /sample
Sample_label_protocol_ch1	0.1M DTT (2.0 µL) /sample
Sample_label_protocol_ch1	10mM dNTPs (1.0 µL) /sample
Sample_label_protocol_ch1	7.0 µL total
Sample_label_protocol_ch1	1. Add 7.0 µL 1st Strand MasterMix to each sample. Mix.
Sample_label_protocol_ch1	2. Put on thermalcycler 42°C-1 Hour. Set timer for 2 minutes.
Sample_label_protocol_ch1	(This setting is actually programmed for 42°C for 1 hour and 3 minutes, to compensate for the extra 2
Sample_label_protocol_ch1	minutes at the beginning before adding the SuperScript II.
Sample_label_protocol_ch1	3. After 2 minutes at 42°C add 1.0 µL of SSII enzyme. (Addition should be done on thermalcycler)
Sample_label_protocol_ch1	4. The rest of the program will run, 1 hour at 42°C.
Sample_label_protocol_ch1	5. Proceed to 2nd Strand.
Sample_label_protocol_ch1	(components should be thawed ~10 minutes before thermalcycler finishes.)
Sample_label_protocol_ch1	Reverse Transcription (RT) 2nd Strand
Sample_label_protocol_ch1	DEPC H20 91.0 µL /sample
Sample_label_protocol_ch1	5X 2nd Strand Mix 30.0 µL /sample
Sample_label_protocol_ch1	E. coli DNA Polymerase I (DNA Pol I) 4.0 µL /sample
Sample_label_protocol_ch1	10mM dNTPs 3.0 µL /sample
Sample_label_protocol_ch1	E. coli DNA ligase (ligase) 1.0 µL /sample
Sample_label_protocol_ch1	RNase H 1.0 µL /sample
Sample_label_protocol_ch1	1. Put samples on thermalcycler, 16°C for 2 hours.
Sample_label_protocol_ch1	2. At 2 hours, add 2.0 µL of T4 DNA Polymerase. Mix.
Sample_label_protocol_ch1	3. Run additional 5 minutes at 16°C.
Sample_label_protocol_ch1	4. Transfer samples to 1.5mL tube with 10.0 µL of 0.5M EDTA. Vortex.
Sample_label_protocol_ch1	5. Use cDNA Clean-Up Module to clean up samples.
Sample_label_protocol_ch1	Clean-Up of Double Stranded cDNA
Sample_label_protocol_ch1	Necessary components:
Sample_label_protocol_ch1	1.5mL tube with cDNA and EDTA, mixed.
Sample_label_protocol_ch1	cDNA Binding Buffer
Sample_label_protocol_ch1	cDNA Wash Buffer (ethanol added by MCF)
Sample_label_protocol_ch1	cDNA Elution Buffer
Sample_label_protocol_ch1	cDNA clean-up column (in 4°C Deli Fridge)
Sample_label_protocol_ch1	Empty Collection Tube (provided in clean-up module)
Sample_label_protocol_ch1	Empty 1.5mL tube labeled with sample name for collecting final eluate.
Sample_label_protocol_ch1	MicroFuge 18 Centrifuge to spin down samples.
Sample_label_protocol_ch1	cDNA Wash Buffer is supplied as a concentrate and you have to add ethanol before using. Check the tube before use to make sure that etOH has been added.
Sample_label_protocol_ch1	1. Label Collection Tube with sample name (on side).
Sample_label_protocol_ch1	2. Add 600.0 µL of cDNA Binding Buffer (yellow) into tube with sample and EDTA.
Sample_label_protocol_ch1	3. Vortex. Spin down briefly (2 sec.)
Sample_label_protocol_ch1	4. Add 500.0 µL sample-Binding Buffer mix onto column.
Sample_label_protocol_ch1	5. Spin in MicroFuge 18 Centrifuge, 14,000rpm for 1 minute.
Sample_label_protocol_ch1	6. Pour eluate down sink. Keep same collection tube.
Sample_label_protocol_ch1	7. Add the remaining cDNA-Binding Buffer mix onto column.
Sample_label_protocol_ch1	8. Spin 14K rpm for 1 minute.
Sample_label_protocol_ch1	9. Discard that collection tube and place column into new, labeled tube.
Sample_label_protocol_ch1	10. Add 720.0 µL cDNA Wash Buffer (ETOH added) to the column.
Sample_label_protocol_ch1	11. Spin 14K rpm for 1 minute.
Sample_label_protocol_ch1	12. Discard eluate into sink. Replace column into collection tube.
Sample_label_protocol_ch1	13. Spin empty column with the lid open for 5 minutes at 14K rpm.
Sample_label_protocol_ch1	14. Place column into labeled 1.5mL tube. Discard collection tube.
Sample_label_protocol_ch1	15. Air dry column for an additional minute (cover with KimWipe).
Sample_label_protocol_ch1	16. Add 14.0 µL Elution Buffer onto the center of the column.
Sample_label_protocol_ch1	17. Let sit at room temp for 1 minute.
Sample_label_protocol_ch1	18. Spin at 14K for 1 minute.
Sample_label_protocol_ch1	19. Discard column.
Sample_label_protocol_ch1	20. Measure the eluate volume.
Sample_label_protocol_ch1	21. Bring volume up to 20.0 µL with DEPC H20.
Sample_label_protocol_ch1	22. Store at -20ºC until ready to proceed with IVT step.
Sample_label_protocol_ch1	InVitro Transcription Reaction (IVT): production of biotin-labeled cRNA
Sample_label_protocol_ch1	cDNA should be at 20.0 µL before adding IVT MasterMix components.
Sample_label_protocol_ch1	IVT kit components should be thawed to room temp. ~20 minutes before using.
Sample_label_protocol_ch1	10X IVT Labeling Buffer (may have precipitate, mix well.) (4.0 µL) /sample
Sample_label_protocol_ch1	IVT Labeling NTP Mix (12.0 µL) /sample
Sample_label_protocol_ch1	IVT Labeling Enzyme Mix (4.0 µL) /sample
Sample_label_protocol_ch1	1. Mix components. Spin briefly.
Sample_label_protocol_ch1	2. Add 20.0 µL IVT MasterMix to each sample. Mix by pipetting.
Sample_label_protocol_ch1	3. 37 ºC for 16 hours in thermalcycler.
Sample_label_protocol_ch1	4. Clean-up cRNA with cRNA Clean-Up Module.
Sample_label_protocol_ch1	Clean-Up of Biotinylated cRNA with cRNA Clean-Up Module
Sample_label_protocol_ch1	Necessary Components:
Sample_label_protocol_ch1	cRNA Binding Buffer
Sample_label_protocol_ch1	cRNA Wash Buffer (ethanol added by MCF)
Sample_label_protocol_ch1	cRNA clean-up column (stored at 4°C in deli fridge)
Sample_label_protocol_ch1	Collection Tube (labeled with sample name on side)
Sample_label_protocol_ch1	DEPC H20
Sample_label_protocol_ch1	100% Ethanol
Sample_label_protocol_ch1	80% Ethanol
Sample_label_protocol_ch1	2 clean, labeled 1.5mL tubes
Sample_label_protocol_ch1	1. Transfer cRNA samples into clean, labeled 1.5mL tubes for clean-up.
Sample_label_protocol_ch1	2. Add 60.0 µL to each sample. Vortex to mix and spin down.
Sample_label_protocol_ch1	3. Add 350.0 µL cRNA Binding Buffer to each sample. Vortex and spin down.
Sample_label_protocol_ch1	4. Add 250.0 µL 100% ETOH to each sample. Mix by pipetting.
Sample_label_protocol_ch1	5. Add cRNA-Binding Buffer-ETOH mix onto column (it all fits).
Sample_label_protocol_ch1	6. Spin at 14,000rpm on “Short Spin” setting (30 seconds).
Sample_label_protocol_ch1	7. Place columns into clean, labeled collection tube. Discard previous tubes with eluate.
Sample_label_protocol_ch1	8. Add 500.0 µL cRNA Wash Buffer to each column.
Sample_label_protocol_ch1	9. Spin at 14K for “Short Spin”.
Sample_label_protocol_ch1	10. Discard eluate in sink but replace column into collection tube.
Sample_label_protocol_ch1	11. Add 500.0 µL 80% ETOH onto column.
Sample_label_protocol_ch1	12. “Short Spin” at 14K.
Sample_label_protocol_ch1	13. Discard eluate and replace columns into collection tube again.
Sample_label_protocol_ch1	14. Spin 14K for 5 minutes to dry column.
Sample_label_protocol_ch1	15. Put column into clean, labeled 1.5mL tube. (This tube should have the request number, the term “IVT” and the sample name on top. On the side put the PI last name and the date.)
Sample_label_protocol_ch1	16. Pipet 11.0 µL DEPC H20 onto center of column.
Sample_label_protocol_ch1	17. Spin 14K for 1 minute.
Sample_label_protocol_ch1	18. Add another 10.0 µL DEPC H20 onto center of column.
Sample_label_protocol_ch1	19. Spin 14K for 1 minute.
Sample_label_protocol_ch1	20. Measure volume of eluate. Record the volume in your lab notebook.
Sample_label_protocol_ch1	21. NanoDrop samples. Dilute with DEPC H20 if you need to, to get concentration into read-able range for NanoDrop (under 3000_g).
Sample_label_protocol_ch1	22. Make a dilution of the IVT samples for testing on the Agilent.
Sample_label_protocol_ch1	Perform calculations to determine adjusted cRNA amount. Enter the total µg into MAID. Perform calculations for Fragmentation step and record the volumes in your lab notebook. Calculations are done with the IVT NanoDrop adjusted cRNA concentrations acquired with the formula provided by Affy.
Sample_label_protocol_ch1	Fragmentation:
Sample_label_protocol_ch1	cRNA (X uL) 15ug
Sample_label_protocol_ch1	DEPC H20(variable) (32-x) µL
Sample_label_protocol_ch1	5X Fragmentation Buffer 8.0 µL
Sample_label_protocol_ch1	40.0 uL
Sample_label_protocol_ch1	1. Add 8.0 µL 5X Frag. Buffer to each empty 1.5mL tube.
Sample_label_protocol_ch1	2. Add the required amount of water to each tube. This amount will depend on the concentration of cRNA- how much cRNA to make 15 µg of fragmented cRNA, then how much DEPC H20 to bring the final volume to 40.0 µL?
Sample_label_protocol_ch1	3. Add the required amount of cRNA to each tube.
Sample_label_protocol_ch1	4. Make sure the 1.5mL tube is clearly labeled FRAG so that you can tell it apart from the IVT cRNA tube.
Sample_label_protocol_ch1	5. Put the FRAG tube onto the 99°C heat block for 35 minutes.
Sample_label_protocol_ch1	6. After fragmentation put the tube on ice. Let it cool about 5 minutes on ice.
Sample_label_protocol_ch1	7. Spin in the MicroFuge at max. speed to get the condensation out of the lid.
Sample_label_protocol_ch1	8. Vortex and spin.
Sample_label_protocol_ch1	9. Test FRAG on NanoDrop. Needs to be 15 µg or more. (15 µg/40 µL=0.375 µg/µL or more)
Sample_label_protocol_ch1	Put the FRAG into hybridization cocktail and store at -20°C until you are ready to load it onto a chip.
Sample_hyb_protocol	Euk 20X must be heated 5 minutes before using it. Vortex before using and spin down. Put on ice when you’re finished with it but not before adding it to MasterMix.
Sample_hyb_protocol	(multiply all by 3.5) MasterMix components
Sample_hyb_protocol	2X Hybe Buffer 150.0 µL 525.0 µL
Sample_hyb_protocol	DEPC H20 54.0 µL (to bring volume up to 300.0µL) 189.0 µL
Sample_hyb_protocol	100% DMSO 30.0 µL 105.0 µL
Sample_hyb_protocol	Eukaryotic 20X Control *** 15.0 µL 52.5 µL
Sample_hyb_protocol	Oligo B2 5.0 µL 17.5 µL
Sample_hyb_protocol	Herring Sperm DNA 3.0 µL 10.5 µL
Sample_hyb_protocol	BSA 3.0 µL 10.5 µL
Sample_hyb_protocol	cRNAfragmented 40.0 µL (if you retrieve all from NanoDrop) you have these separate
Sample_hyb_protocol	1. Keep cRNA in tubes labeled FRAG.
Sample_hyb_protocol	2. Prepare MasterMix in 15mL corning tube.
Sample_hyb_protocol	3. Mix after each component by pipetting several times.
Sample_hyb_protocol	4. Add 260.0 µL of the Hybe MasterMix to each fragmented sample. You total volume should be 300.0 µL.
Sample_hyb_protocol	5. If you are not hybridizing right away, store the cRNA-HybeCocktail mix in the -20°C until you’re ready to use it.
Sample_hyb_protocol	HYBRIDIZATION
Sample_hyb_protocol	Thaw Hybe Cocktails if they are frozen before heating. Pull arrays out of 4°C Deli Fridge 30 minutes before you want to load them. They must be at room temperature before you pierce the septa to prevent leakage.
Sample_hyb_protocol	1. Put tube hats on to prevent popping open at high heat.
Sample_hyb_protocol	2. Place the tubes into the 95°C heating block for 5 minutes.
Sample_hyb_protocol	3. Move the tubes to the 45°C heating block, again for 5 minutes.
Sample_hyb_protocol	4. Spin in MicroFuge at max. speed for 5 minutes.
Sample_hyb_protocol	5. Load 250.0 µL into each array.
Sample_hyb_protocol	6. Put in Hybe Oven, set at 60rpm and 45°C for 16 hours.
Sample_hyb_protocol	After 16 hours of hybridization, remove the hybridization cocktail from the probe array and fill the probe array completely with the appropriate volume of Non-Stringent Wash Buffer (Wash Buffer A)
Sample_hyb_protocol	Wash Buffer A: Non-Stringent Wash Buffer
Sample_hyb_protocol	(6X SSPE, 0.01% Tween-20)
Sample_hyb_protocol	For 1,000 mL:
Sample_hyb_protocol	300 mL of 20X SSPE
Sample_hyb_protocol	1.0 mL of 10% Tween-20
Sample_hyb_protocol	699 mL of water
Sample_hyb_protocol	Filter through a 0.2 μm filter
Sample_hyb_protocol	EukGE-WS2v5*
Sample_hyb_protocol	Midi_euk2*
Sample_hyb_protocol	10 cycles of 2 mixes/cycle with Wash
Sample_hyb_protocol	Buffer A at 30°C 6 cycles of 15 mixes/cycle with
Sample_hyb_protocol	Wash Buffer B at 50°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in SAPE solution at 35°C
Sample_hyb_protocol	10 cycles of 4 mixes/cycle with Wash Buffer A at 30°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in antibody solution at 35°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in SAPE solution at 35°C
Sample_hyb_protocol	15 cycles of 4 mixes/cycle with Wash Buffer A at 35°C. The holding temperature is 25°C
Sample_scan_protocol	[Scanner]
Sample_scan_protocol	Pixel Size 1.56
Sample_scan_protocol	hardware: GeneChip Scanner 3000
Sample_scan_protocol	Affymetrix® Microarray Suite
Sample_scan_protocol	Filter 570
Sample_scan_protocol	Scanner ID 50101490
Sample_scan_protocol	Number of Scans 1
Sample_scan_protocol	Scanner Type M10
Sample_scan_protocol	As affymetrix protocol.
Sample_data_processing	Data in value is raw data (Mas5 scaled) unaltered by normalizations
Sample_platform_id	GPL1261
Sample_contact_name	Xiangyin,,kong
Sample_contact_email	xykong@sibs.ac.cn
Sample_contact_laboratory	molecular genetics
Sample_contact_department	molecular genetics
Sample_contact_institute	Institute of health science
Sample_contact_address	No.225 yueyang road
Sample_contact_city	Shanghai
Sample_contact_zip/postal_code	200031
Sample_contact_country	China
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM312nnn/GSM312177/suppl/GSM312177.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM312nnn/GSM312177/suppl/GSM312177.CHP.gz
Sample_series_id	GSE12415
Sample_data_row_count	45101

GSM312196

GPL1261

HSF4 knockout2

Postnatal Day 0.5 lens tissue, HSF4 knockout

Background Srain: 129S3 Gender: Male Tissue: lens Age: 0.5 Days Postnatal

sxh ko2

Sample_geo_accession	GSM312196
Sample_status	Public on Aug 11 2009
Sample_submission_date	Aug 13 2008
Sample_last_update_date	Aug 18 2008
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_treatment_protocol_ch1	Lens were dissected from day 0.5 animals and placed in RNAlater (Ambion) for 24 hours at 4 degrees Celsius. After 24 hours, the RNAlater was removed and the lens were frozen on dry ice and placed in a -80 degree freezer.
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	RNA was isolated using Qiagen RNeasy mini kits. On column DNase digestion was used. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Affymatrix Bioanalyzer system.
Sample_label_ch1	Biotin
Sample_label_protocol_ch1 = Test RNA sample on NanoDrop. Concentration must be 500_g/µL or more. Calculate the volume of RNA sample necessary to start protocol with 5µg. i.e. (5 µg) / (NanoDrop concentration reading in µg/µL)	µL
Sample_label_protocol_ch1	RNA x uL
Sample_label_protocol_ch1	DEPC H20 10-x uL
Sample_label_protocol_ch1	oligo T7 2.0 µL
Sample_label_protocol_ch1	1. Add all components together in a 0.2µL strip tube.
Sample_label_protocol_ch1	2. Mix well and spin down.
Sample_label_protocol_ch1	3. Place in thermalcycler at 70°C for 10 minutes. Should be warming up to 70°C while you prep. the samples.
Sample_label_protocol_ch1	4. Allow thermalcycler to ramp down to 4°C.
Sample_label_protocol_ch1	5. 4°C for 2 minutes. Spin down and put on ice.
Sample_label_protocol_ch1	6. Prepare the 1st Strand MasterMix.
Sample_label_protocol_ch1	Reverse Transcription (RT) 1st Strand
Sample_label_protocol_ch1	You should have thawed and vortexed the components during the 70°C-10 minute step.
Sample_label_protocol_ch1	5X 1st Strand Mix (4.0 µL) /sample
Sample_label_protocol_ch1	0.1M DTT (2.0 µL) /sample
Sample_label_protocol_ch1	10mM dNTPs (1.0 µL) /sample
Sample_label_protocol_ch1	7.0 µL total
Sample_label_protocol_ch1	1. Add 7.0 µL 1st Strand MasterMix to each sample. Mix.
Sample_label_protocol_ch1	2. Put on thermalcycler 42°C-1 Hour. Set timer for 2 minutes.
Sample_label_protocol_ch1	(This setting is actually programmed for 42°C for 1 hour and 3 minutes, to compensate for the extra 2
Sample_label_protocol_ch1	minutes at the beginning before adding the SuperScript II.
Sample_label_protocol_ch1	3. After 2 minutes at 42°C add 1.0 µL of SSII enzyme. (Addition should be done on thermalcycler)
Sample_label_protocol_ch1	4. The rest of the program will run, 1 hour at 42°C.
Sample_label_protocol_ch1	5. Proceed to 2nd Strand.
Sample_label_protocol_ch1	(components should be thawed ~10 minutes before thermalcycler finishes.)
Sample_label_protocol_ch1	Reverse Transcription (RT) 2nd Strand
Sample_label_protocol_ch1	DEPC H20 91.0 µL /sample
Sample_label_protocol_ch1	5X 2nd Strand Mix 30.0 µL /sample
Sample_label_protocol_ch1	E. coli DNA Polymerase I (DNA Pol I) 4.0 µL /sample
Sample_label_protocol_ch1	10mM dNTPs 3.0 µL /sample
Sample_label_protocol_ch1	E. coli DNA ligase (ligase) 1.0 µL /sample
Sample_label_protocol_ch1	RNase H 1.0 µL /sample
Sample_label_protocol_ch1	1. Put samples on thermalcycler, 16°C for 2 hours.
Sample_label_protocol_ch1	2. At 2 hours, add 2.0 µL of T4 DNA Polymerase. Mix.
Sample_label_protocol_ch1	3. Run additional 5 minutes at 16°C.
Sample_label_protocol_ch1	4. Transfer samples to 1.5mL tube with 10.0 µL of 0.5M EDTA. Vortex.
Sample_label_protocol_ch1	5. Use cDNA Clean-Up Module to clean up samples.
Sample_label_protocol_ch1	Clean-Up of Double Stranded cDNA
Sample_label_protocol_ch1	Necessary components:
Sample_label_protocol_ch1	1.5mL tube with cDNA and EDTA, mixed.
Sample_label_protocol_ch1	cDNA Binding Buffer
Sample_label_protocol_ch1	cDNA Wash Buffer (ethanol added by MCF)
Sample_label_protocol_ch1	cDNA Elution Buffer
Sample_label_protocol_ch1	cDNA clean-up column (in 4°C Deli Fridge)
Sample_label_protocol_ch1	Empty Collection Tube (provided in clean-up module)
Sample_label_protocol_ch1	Empty 1.5mL tube labeled with sample name for collecting final eluate.
Sample_label_protocol_ch1	MicroFuge 18 Centrifuge to spin down samples.
Sample_label_protocol_ch1	cDNA Wash Buffer is supplied as a concentrate and you have to add ethanol before using. Check the tube before use to make sure that etOH has been added.
Sample_label_protocol_ch1	1. Label Collection Tube with sample name (on side).
Sample_label_protocol_ch1	2. Add 600.0 µL of cDNA Binding Buffer (yellow) into tube with sample and EDTA.
Sample_label_protocol_ch1	3. Vortex. Spin down briefly (2 sec.)
Sample_label_protocol_ch1	4. Add 500.0 µL sample-Binding Buffer mix onto column.
Sample_label_protocol_ch1	5. Spin in MicroFuge 18 Centrifuge, 14,000rpm for 1 minute.
Sample_label_protocol_ch1	6. Pour eluate down sink. Keep same collection tube.
Sample_label_protocol_ch1	7. Add the remaining cDNA-Binding Buffer mix onto column.
Sample_label_protocol_ch1	8. Spin 14K rpm for 1 minute.
Sample_label_protocol_ch1	9. Discard that collection tube and place column into new, labeled tube.
Sample_label_protocol_ch1	10. Add 720.0 µL cDNA Wash Buffer (ETOH added) to the column.
Sample_label_protocol_ch1	11. Spin 14K rpm for 1 minute.
Sample_label_protocol_ch1	12. Discard eluate into sink. Replace column into collection tube.
Sample_label_protocol_ch1	13. Spin empty column with the lid open for 5 minutes at 14K rpm.
Sample_label_protocol_ch1	14. Place column into labeled 1.5mL tube. Discard collection tube.
Sample_label_protocol_ch1	15. Air dry column for an additional minute (cover with KimWipe).
Sample_label_protocol_ch1	16. Add 14.0 µL Elution Buffer onto the center of the column.
Sample_label_protocol_ch1	17. Let sit at room temp for 1 minute.
Sample_label_protocol_ch1	18. Spin at 14K for 1 minute.
Sample_label_protocol_ch1	19. Discard column.
Sample_label_protocol_ch1	20. Measure the eluate volume.
Sample_label_protocol_ch1	21. Bring volume up to 20.0 µL with DEPC H20.
Sample_label_protocol_ch1	22. Store at -20ºC until ready to proceed with IVT step.
Sample_label_protocol_ch1	InVitro Transcription Reaction (IVT): production of biotin-labeled cRNA
Sample_label_protocol_ch1	cDNA should be at 20.0 µL before adding IVT MasterMix components.
Sample_label_protocol_ch1	IVT kit components should be thawed to room temp. ~20 minutes before using.
Sample_label_protocol_ch1	10X IVT Labeling Buffer (may have precipitate, mix well.) (4.0 µL) /sample
Sample_label_protocol_ch1	IVT Labeling NTP Mix (12.0 µL) /sample
Sample_label_protocol_ch1	IVT Labeling Enzyme Mix (4.0 µL) /sample
Sample_label_protocol_ch1	1. Mix components. Spin briefly.
Sample_label_protocol_ch1	2. Add 20.0 µL IVT MasterMix to each sample. Mix by pipetting.
Sample_label_protocol_ch1	3. 37 ºC for 16 hours in thermalcycler.
Sample_label_protocol_ch1	4. Clean-up cRNA with cRNA Clean-Up Module.
Sample_label_protocol_ch1	Clean-Up of Biotinylated cRNA with cRNA Clean-Up Module
Sample_label_protocol_ch1	Necessary Components:
Sample_label_protocol_ch1	cRNA Binding Buffer
Sample_label_protocol_ch1	cRNA Wash Buffer (ethanol added by MCF)
Sample_label_protocol_ch1	cRNA clean-up column (stored at 4°C in deli fridge)
Sample_label_protocol_ch1	Collection Tube (labeled with sample name on side)
Sample_label_protocol_ch1	DEPC H20
Sample_label_protocol_ch1	100% Ethanol
Sample_label_protocol_ch1	80% Ethanol
Sample_label_protocol_ch1	2 clean, labeled 1.5mL tubes
Sample_label_protocol_ch1	1. Transfer cRNA samples into clean, labeled 1.5mL tubes for clean-up.
Sample_label_protocol_ch1	2. Add 60.0 µL to each sample. Vortex to mix and spin down.
Sample_label_protocol_ch1	3. Add 350.0 µL cRNA Binding Buffer to each sample. Vortex and spin down.
Sample_label_protocol_ch1	4. Add 250.0 µL 100% ETOH to each sample. Mix by pipetting.
Sample_label_protocol_ch1	5. Add cRNA-Binding Buffer-ETOH mix onto column (it all fits).
Sample_label_protocol_ch1	6. Spin at 14,000rpm on “Short Spin” setting (30 seconds).
Sample_label_protocol_ch1	7. Place columns into clean, labeled collection tube. Discard previous tubes with eluate.
Sample_label_protocol_ch1	8. Add 500.0 µL cRNA Wash Buffer to each column.
Sample_label_protocol_ch1	9. Spin at 14K for “Short Spin”.
Sample_label_protocol_ch1	10. Discard eluate in sink but replace column into collection tube.
Sample_label_protocol_ch1	11. Add 500.0 µL 80% ETOH onto column.
Sample_label_protocol_ch1	12. “Short Spin” at 14K.
Sample_label_protocol_ch1	13. Discard eluate and replace columns into collection tube again.
Sample_label_protocol_ch1	14. Spin 14K for 5 minutes to dry column.
Sample_label_protocol_ch1	15. Put column into clean, labeled 1.5mL tube. (This tube should have the request number, the term “IVT” and the sample name on top. On the side put the PI last name and the date.)
Sample_label_protocol_ch1	16. Pipet 11.0 µL DEPC H20 onto center of column.
Sample_label_protocol_ch1	17. Spin 14K for 1 minute.
Sample_label_protocol_ch1	18. Add another 10.0 µL DEPC H20 onto center of column.
Sample_label_protocol_ch1	19. Spin 14K for 1 minute.
Sample_label_protocol_ch1	20. Measure volume of eluate. Record the volume in your lab notebook.
Sample_label_protocol_ch1	21. NanoDrop samples. Dilute with DEPC H20 if you need to, to get concentration into read-able range for NanoDrop (under 3000_g).
Sample_label_protocol_ch1	22. Make a dilution of the IVT samples for testing on the Agilent.
Sample_label_protocol_ch1	Perform calculations to determine adjusted cRNA amount. Enter the total µg into MAID. Perform calculations for Fragmentation step and record the volumes in your lab notebook. Calculations are done with the IVT NanoDrop adjusted cRNA concentrations acquired with the formula provided by Affy.
Sample_label_protocol_ch1	Fragmentation:
Sample_label_protocol_ch1	cRNA (X uL) 15ug
Sample_label_protocol_ch1	DEPC H20(variable) (32-x) µL
Sample_label_protocol_ch1	5X Fragmentation Buffer 8.0 µL
Sample_label_protocol_ch1	40.0 uL
Sample_label_protocol_ch1	1. Add 8.0 µL 5X Frag. Buffer to each empty 1.5mL tube.
Sample_label_protocol_ch1	2. Add the required amount of water to each tube. This amount will depend on the concentration of cRNA- how much cRNA to make 15 µg of fragmented cRNA, then how much DEPC H20 to bring the final volume to 40.0 µL?
Sample_label_protocol_ch1	3. Add the required amount of cRNA to each tube.
Sample_label_protocol_ch1	4. Make sure the 1.5mL tube is clearly labeled FRAG so that you can tell it apart from the IVT cRNA tube.
Sample_label_protocol_ch1	5. Put the FRAG tube onto the 99°C heat block for 35 minutes.
Sample_label_protocol_ch1	6. After fragmentation put the tube on ice. Let it cool about 5 minutes on ice.
Sample_label_protocol_ch1	7. Spin in the MicroFuge at max. speed to get the condensation out of the lid.
Sample_label_protocol_ch1	8. Vortex and spin.
Sample_label_protocol_ch1	9. Test FRAG on NanoDrop. Needs to be 15 µg or more. (15 µg/40 µL=0.375 µg/µL or more)
Sample_label_protocol_ch1	Put the FRAG into hybridization cocktail and store at -20°C until you are ready to load it onto a chip.
Sample_hyb_protocol	Euk 20X must be heated 5 minutes before using it. Vortex before using and spin down. Put on ice when you’re finished with it but not before adding it to MasterMix.
Sample_hyb_protocol	(multiply all by 3.5) MasterMix components
Sample_hyb_protocol	2X Hybe Buffer 150.0 µL 525.0 µL
Sample_hyb_protocol	DEPC H20 54.0 µL (to bring volume up to 300.0µL) 189.0 µL
Sample_hyb_protocol	100% DMSO 30.0 µL 105.0 µL
Sample_hyb_protocol	Eukaryotic 20X Control *** 15.0 µL 52.5 µL
Sample_hyb_protocol	Oligo B2 5.0 µL 17.5 µL
Sample_hyb_protocol	Herring Sperm DNA 3.0 µL 10.5 µL
Sample_hyb_protocol	BSA 3.0 µL 10.5 µL
Sample_hyb_protocol	cRNAfragmented 40.0 µL (if you retrieve all from NanoDrop) you have these separate
Sample_hyb_protocol	1. Keep cRNA in tubes labeled FRAG.
Sample_hyb_protocol	2. Prepare MasterMix in 15mL corning tube.
Sample_hyb_protocol	3. Mix after each component by pipetting several times.
Sample_hyb_protocol	4. Add 260.0 µL of the Hybe MasterMix to each fragmented sample. You total volume should be 300.0 µL.
Sample_hyb_protocol	5. If you are not hybridizing right away, store the cRNA-HybeCocktail mix in the -20°C until you’re ready to use it.
Sample_hyb_protocol	HYBRIDIZATION
Sample_hyb_protocol	Thaw Hybe Cocktails if they are frozen before heating. Pull arrays out of 4°C Deli Fridge 30 minutes before you want to load them. They must be at room temperature before you pierce the septa to prevent leakage.
Sample_hyb_protocol	1. Put tube hats on to prevent popping open at high heat.
Sample_hyb_protocol	2. Place the tubes into the 95°C heating block for 5 minutes.
Sample_hyb_protocol	3. Move the tubes to the 45°C heating block, again for 5 minutes.
Sample_hyb_protocol	4. Spin in MicroFuge at max. speed for 5 minutes.
Sample_hyb_protocol	5. Load 250.0 µL into each array.
Sample_hyb_protocol	6. Put in Hybe Oven, set at 60rpm and 45°C for 16 hours.
Sample_hyb_protocol	After 16 hours of hybridization, remove the hybridization cocktail from the probe array and fill the probe array completely with the appropriate volume of Non-Stringent Wash Buffer (Wash Buffer A)
Sample_hyb_protocol	Wash Buffer A: Non-Stringent Wash Buffer
Sample_hyb_protocol	(6X SSPE, 0.01% Tween-20)
Sample_hyb_protocol	For 1,000 mL:
Sample_hyb_protocol	300 mL of 20X SSPE
Sample_hyb_protocol	1.0 mL of 10% Tween-20
Sample_hyb_protocol	699 mL of water
Sample_hyb_protocol	Filter through a 0.2 μm filter
Sample_hyb_protocol	EukGE-WS2v5*
Sample_hyb_protocol	Midi_euk2*
Sample_hyb_protocol	10 cycles of 2 mixes/cycle with Wash
Sample_hyb_protocol	Buffer A at 30°C 6 cycles of 15 mixes/cycle with
Sample_hyb_protocol	Wash Buffer B at 50°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in SAPE solution at 35°C
Sample_hyb_protocol	10 cycles of 4 mixes/cycle with Wash Buffer A at 30°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in antibody solution at 35°C
Sample_hyb_protocol	Stain the probe array for 5 minutes in SAPE solution at 35°C
Sample_hyb_protocol	15 cycles of 4 mixes/cycle with Wash Buffer A at 35°C. The holding temperature is 25°C
Sample_scan_protocol	[Scanner]
Sample_scan_protocol	Pixel Size 1.56
Sample_scan_protocol	hardware: GeneChip Scanner 3000
Sample_scan_protocol	Affymetrix® Microarray Suite
Sample_scan_protocol	Filter 570
Sample_scan_protocol	Scanner ID 50101490
Sample_scan_protocol	Number of Scans 1
Sample_scan_protocol	Scanner Type M10
Sample_scan_protocol	As affymetrix protocol.
Sample_data_processing	Data in value is raw data (Mas5 scaled) unaltered by normalizations
Sample_platform_id	GPL1261
Sample_contact_name	Xiangyin,,kong
Sample_contact_email	xykong@sibs.ac.cn
Sample_contact_laboratory	molecular genetics
Sample_contact_department	molecular genetics
Sample_contact_institute	Institute of health science
Sample_contact_address	No.225 yueyang road
Sample_contact_city	Shanghai
Sample_contact_zip/postal_code	200031
Sample_contact_country	China
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM312nnn/GSM312196/suppl/GSM312196.CEL.gz
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM312nnn/GSM312196/suppl/GSM312196.CHP.gz
Sample_series_id	GSE12415
Sample_data_row_count	45101

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