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GSM358295

GPL1261

E13.5 Mouse testis_129/Sv

E13.5 male genital ridge 129/Sv

Background Srain: 129/SV Gender: male Tissue: testis Age: E13.5

gene expression differences between 129/Sv and 129.MOLF-Chr19 strains

Sample_geo_accession	GSM358295
Sample_status	Public on Jan 13 2009
Sample_submission_date	Jan 07 2009
Sample_last_update_date	Jan 12 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	20 pairs of testes were pooled. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
Sample_label_protocol_ch1	Use a starting amount of 5.0 – 40.0 mg total RNA.
Sample_label_protocol_ch1	First Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
Sample_label_protocol_ch1	2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
Sample_label_protocol_ch1	4.Incubate at 42° C for 2 minutes.
Sample_label_protocol_ch1	5.Add to the tube 1 mL of Superscript II reverse transcriptase.
Sample_label_protocol_ch1	6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
Sample_label_protocol_ch1	2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
Sample_label_protocol_ch1	3.Add 2 mL [10 U] T4 DNA Polymerase.
Sample_label_protocol_ch1	4.Incubate at 16° C for 5 minutes.
Sample_label_protocol_ch1	5.Add 10 mL 0.5 M EDTA.
Sample_label_protocol_ch1	Cleanup of Double-Stranded cDNA
Sample_label_protocol_ch1	Phenol/Chloroform Extraction
Sample_label_protocol_ch1	1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
Sample_label_protocol_ch1	2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
Sample_label_protocol_ch1	Ethanol Precipitation
Sample_label_protocol_ch1	1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
Sample_label_protocol_ch1	2.Immediately spin at maximum speed for 20 minutes at room temperature.
Sample_label_protocol_ch1	3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	5.Air dry pellet.
Sample_label_protocol_ch1	6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
Sample_label_protocol_ch1	Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
Sample_label_protocol_ch1	IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
Sample_label_protocol_ch1	1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
Sample_label_protocol_ch1	2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
Sample_label_protocol_ch1	IVT Cleanup
Sample_label_protocol_ch1	-The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
Sample_label_protocol_ch1	-The RNA elution step is performed twice to increase recovery.
Sample_label_protocol_ch1	-Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
Sample_label_protocol_ch1	Quantifying the cRNA
Sample_label_protocol_ch1	1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
Sample_label_protocol_ch1	2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
Sample_label_protocol_ch1	3.The following formula is used to determine the cRNA yield.
Sample_label_protocol_ch1	Adjusted cRNA yield=RNA* - (total RNA#)($)
Sample_label_protocol_ch1 = RNA*	amount of cRNA measured after IVT (mg)
Sample_label_protocol_ch1 = total RNA#	sterting amount of total RNA (mg)
Sample_label_protocol_ch1 = $	fraction of cRNA reaction used in IVT
Sample_label_protocol_ch1	cRNA Fragmentation
Sample_label_protocol_ch1	1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
Sample_label_protocol_ch1	2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
Sample_label_protocol_ch1	3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
Sample_label_protocol_ch1	Gel Electrophoresis
Sample_label_protocol_ch1	1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
Sample_label_protocol_ch1	2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
Sample_hyb_protocol	1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
Sample_hyb_protocol	2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
Sample_hyb_protocol	3.Prepare the hybridization cocktail as follows:
Sample_hyb_protocol	Reagents Mini Array Midi Array Standard Array
Sample_hyb_protocol	Fragmented cRNA 5mg 10mg 15 mg
Sample_hyb_protocol	Control oligo B2 1.7ml 3.3ml 5ml
Sample_hyb_protocol	20X eukaryotic hybridization controls 5ml 10ml 15ml
Sample_hyb_protocol	Herring sperm DNA 1ml 2ml 3ml
Sample_hyb_protocol	Acetylated BSA 1ml 2ml 3ml
Sample_hyb_protocol	2X Hybridization buffer 50ml 100ml 150mul
Sample_hyb_protocol	Water to 100ml to 200ml to 300ml
Sample_hyb_protocol	4.Equilibrate probe array to room temperature.
Sample_hyb_protocol	5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
Sample_hyb_protocol	6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
Sample_hyb_protocol	7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
Sample_hyb_protocol	8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
Sample_hyb_protocol	Washing and Staining Probe Arrays
Sample_hyb_protocol	Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
Sample_scan_protocol	The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
Sample_data_processing	dChip (version 1.3) scaled. Normalized.
Sample_platform_id	GPL1261
Sample_contact_name	Rui,,Zhu
Sample_contact_email	rzhu@mdanderson.org
Sample_contact_laboratory	Matin
Sample_contact_department	Genetics
Sample_contact_institute	UT MD Anderson Cancer Center
Sample_contact_address	1515 Holcombe Blvd.
Sample_contact_city	Houston
Sample_contact_state	TX
Sample_contact_zip/postal_code	77030
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM358nnn/GSM358295/suppl/GSM358295.CEL.gz
Sample_series_id	GSE14354
Sample_data_row_count	45101

GSM358296

GPL1261

E13.5 Mouse testis_M19

E13.5 male genital ridges, 129.MOLF-Chr 19

Background Srain: 129.MOLF-Chr 19 Gender: male Tissue: testis Age: E13.5

gene expression differences between 129/Sv and 129.MOLF-Chr19 strains

Sample_geo_accession	GSM358296
Sample_status	Public on Jan 13 2009
Sample_submission_date	Jan 07 2009
Sample_last_update_date	Jan 12 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	20 pairs of testes were pooled. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
Sample_label_protocol_ch1	Use a starting amount of 5.0 – 40.0 mg total RNA.
Sample_label_protocol_ch1	First Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
Sample_label_protocol_ch1	2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
Sample_label_protocol_ch1	4.Incubate at 42° C for 2 minutes.
Sample_label_protocol_ch1	5.Add to the tube 1 mL of Superscript II reverse transcriptase.
Sample_label_protocol_ch1	6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
Sample_label_protocol_ch1	2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
Sample_label_protocol_ch1	3.Add 2 mL [10 U] T4 DNA Polymerase.
Sample_label_protocol_ch1	4.Incubate at 16° C for 5 minutes.
Sample_label_protocol_ch1	5.Add 10 mL 0.5 M EDTA.
Sample_label_protocol_ch1	Cleanup of Double-Stranded cDNA
Sample_label_protocol_ch1	Phenol/Chloroform Extraction
Sample_label_protocol_ch1	1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
Sample_label_protocol_ch1	2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
Sample_label_protocol_ch1	Ethanol Precipitation
Sample_label_protocol_ch1	1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
Sample_label_protocol_ch1	2.Immediately spin at maximum speed for 20 minutes at room temperature.
Sample_label_protocol_ch1	3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	5.Air dry pellet.
Sample_label_protocol_ch1	6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
Sample_label_protocol_ch1	Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
Sample_label_protocol_ch1	IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
Sample_label_protocol_ch1	1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
Sample_label_protocol_ch1	2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
Sample_label_protocol_ch1	IVT Cleanup
Sample_label_protocol_ch1	-The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
Sample_label_protocol_ch1	-The RNA elution step is performed twice to increase recovery.
Sample_label_protocol_ch1	-Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
Sample_label_protocol_ch1	Quantifying the cRNA
Sample_label_protocol_ch1	1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
Sample_label_protocol_ch1	2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
Sample_label_protocol_ch1	3.The following formula is used to determine the cRNA yield.
Sample_label_protocol_ch1	Adjusted cRNA yield=RNA* - (total RNA#)($)
Sample_label_protocol_ch1 = RNA*	amount of cRNA measured after IVT (mg)
Sample_label_protocol_ch1 = total RNA#	sterting amount of total RNA (mg)
Sample_label_protocol_ch1 = $	fraction of cRNA reaction used in IVT
Sample_label_protocol_ch1	cRNA Fragmentation
Sample_label_protocol_ch1	1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
Sample_label_protocol_ch1	2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
Sample_label_protocol_ch1	3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
Sample_label_protocol_ch1	Gel Electrophoresis
Sample_label_protocol_ch1	1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
Sample_label_protocol_ch1	2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
Sample_hyb_protocol	1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
Sample_hyb_protocol	2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
Sample_hyb_protocol	3.Prepare the hybridization cocktail as follows:
Sample_hyb_protocol	Reagents Mini Array Midi Array Standard Array
Sample_hyb_protocol	Fragmented cRNA 5mg 10mg 15 mg
Sample_hyb_protocol	Control oligo B2 1.7ml 3.3ml 5ml
Sample_hyb_protocol	20X eukaryotic hybridization controls 5ml 10ml 15ml
Sample_hyb_protocol	Herring sperm DNA 1ml 2ml 3ml
Sample_hyb_protocol	Acetylated BSA 1ml 2ml 3ml
Sample_hyb_protocol	2X Hybridization buffer 50ml 100ml 150mul
Sample_hyb_protocol	Water to 100ml to 200ml to 300ml
Sample_hyb_protocol	4.Equilibrate probe array to room temperature.
Sample_hyb_protocol	5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
Sample_hyb_protocol	6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
Sample_hyb_protocol	7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
Sample_hyb_protocol	8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
Sample_hyb_protocol	Washing and Staining Probe Arrays
Sample_hyb_protocol	Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
Sample_scan_protocol	The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
Sample_data_processing	dChip (version 1.3) scaled. Normalized.
Sample_platform_id	GPL1261
Sample_contact_name	Rui,,Zhu
Sample_contact_email	rzhu@mdanderson.org
Sample_contact_laboratory	Matin
Sample_contact_department	Genetics
Sample_contact_institute	UT MD Anderson Cancer Center
Sample_contact_address	1515 Holcombe Blvd.
Sample_contact_city	Houston
Sample_contact_state	TX
Sample_contact_zip/postal_code	77030
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM358nnn/GSM358296/suppl/GSM358296.CEL.gz
Sample_series_id	GSE14354
Sample_data_row_count	45101

GSM358297

GPL1261

E13.5 male embryos_129/Sv

E13.5 male embryos 129/Sv

Background Srain: 129/SV Gender: male Tissue: whole embryos Age: E13.5

gene expression differences between 129/Sv and 129.MOLF-Chr19 strains

Sample_geo_accession	GSM358297
Sample_status	Public on Jan 13 2009
Sample_submission_date	Jan 07 2009
Sample_last_update_date	Jan 12 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	3 male embryos were pooled. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
Sample_label_protocol_ch1	Use a starting amount of 5.0 – 40.0 mg total RNA.
Sample_label_protocol_ch1	First Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
Sample_label_protocol_ch1	2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
Sample_label_protocol_ch1	4.Incubate at 42° C for 2 minutes.
Sample_label_protocol_ch1	5.Add to the tube 1 mL of Superscript II reverse transcriptase.
Sample_label_protocol_ch1	6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
Sample_label_protocol_ch1	2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
Sample_label_protocol_ch1	3.Add 2 mL [10 U] T4 DNA Polymerase.
Sample_label_protocol_ch1	4.Incubate at 16° C for 5 minutes.
Sample_label_protocol_ch1	5.Add 10 mL 0.5 M EDTA.
Sample_label_protocol_ch1	Cleanup of Double-Stranded cDNA
Sample_label_protocol_ch1	Phenol/Chloroform Extraction
Sample_label_protocol_ch1	1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
Sample_label_protocol_ch1	2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
Sample_label_protocol_ch1	Ethanol Precipitation
Sample_label_protocol_ch1	1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
Sample_label_protocol_ch1	2.Immediately spin at maximum speed for 20 minutes at room temperature.
Sample_label_protocol_ch1	3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	5.Air dry pellet.
Sample_label_protocol_ch1	6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
Sample_label_protocol_ch1	Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
Sample_label_protocol_ch1	IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
Sample_label_protocol_ch1	1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
Sample_label_protocol_ch1	2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
Sample_label_protocol_ch1	IVT Cleanup
Sample_label_protocol_ch1	-The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
Sample_label_protocol_ch1	-The RNA elution step is performed twice to increase recovery.
Sample_label_protocol_ch1	-Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
Sample_label_protocol_ch1	Quantifying the cRNA
Sample_label_protocol_ch1	1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
Sample_label_protocol_ch1	2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
Sample_label_protocol_ch1	3.The following formula is used to determine the cRNA yield.
Sample_label_protocol_ch1	Adjusted cRNA yield=RNA* - (total RNA#)($)
Sample_label_protocol_ch1 = RNA*	amount of cRNA measured after IVT (mg)
Sample_label_protocol_ch1 = total RNA#	sterting amount of total RNA (mg)
Sample_label_protocol_ch1 = $	fraction of cRNA reaction used in IVT
Sample_label_protocol_ch1	cRNA Fragmentation
Sample_label_protocol_ch1	1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
Sample_label_protocol_ch1	2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
Sample_label_protocol_ch1	3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
Sample_label_protocol_ch1	Gel Electrophoresis
Sample_label_protocol_ch1	1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
Sample_label_protocol_ch1	2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
Sample_hyb_protocol	1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
Sample_hyb_protocol	2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
Sample_hyb_protocol	3.Prepare the hybridization cocktail as follows:
Sample_hyb_protocol	Reagents Mini Array Midi Array Standard Array
Sample_hyb_protocol	Fragmented cRNA 5mg 10mg 15 mg
Sample_hyb_protocol	Control oligo B2 1.7ml 3.3ml 5ml
Sample_hyb_protocol	20X eukaryotic hybridization controls 5ml 10ml 15ml
Sample_hyb_protocol	Herring sperm DNA 1ml 2ml 3ml
Sample_hyb_protocol	Acetylated BSA 1ml 2ml 3ml
Sample_hyb_protocol	2X Hybridization buffer 50ml 100ml 150mul
Sample_hyb_protocol	Water to 100ml to 200ml to 300ml
Sample_hyb_protocol	4.Equilibrate probe array to room temperature.
Sample_hyb_protocol	5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
Sample_hyb_protocol	6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
Sample_hyb_protocol	7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
Sample_hyb_protocol	8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
Sample_hyb_protocol	Washing and Staining Probe Arrays
Sample_hyb_protocol	Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
Sample_scan_protocol	The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
Sample_data_processing	dChip (version 1.3) scaled. Normalized.
Sample_platform_id	GPL1261
Sample_contact_name	Rui,,Zhu
Sample_contact_email	rzhu@mdanderson.org
Sample_contact_laboratory	Matin
Sample_contact_department	Genetics
Sample_contact_institute	UT MD Anderson Cancer Center
Sample_contact_address	1515 Holcombe Blvd.
Sample_contact_city	Houston
Sample_contact_state	TX
Sample_contact_zip/postal_code	77030
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM358nnn/GSM358297/suppl/GSM358297.CEL.gz
Sample_series_id	GSE14354
Sample_data_row_count	45101

GSM358306

GPL1261

E13.5 male embryos_M19

E13.5 male embryos 129.MOLF-Chr19

Background Srain: 129.MOLF-Chr 19 Gender: male Tissue: whole embryos Age: E13.5

gene expression differences between 129/Sv and 129.MOLF-Chr19 strains

Sample_geo_accession	GSM358306
Sample_status	Public on Jan 13 2009
Sample_submission_date	Jan 07 2009
Sample_last_update_date	Jan 12 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	3 male embryos were pooled. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
Sample_label_protocol_ch1	Use a starting amount of 5.0 – 40.0 mg total RNA.
Sample_label_protocol_ch1	First Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
Sample_label_protocol_ch1	2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
Sample_label_protocol_ch1	4.Incubate at 42° C for 2 minutes.
Sample_label_protocol_ch1	5.Add to the tube 1 mL of Superscript II reverse transcriptase.
Sample_label_protocol_ch1	6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
Sample_label_protocol_ch1	2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
Sample_label_protocol_ch1	3.Add 2 mL [10 U] T4 DNA Polymerase.
Sample_label_protocol_ch1	4.Incubate at 16° C for 5 minutes.
Sample_label_protocol_ch1	5.Add 10 mL 0.5 M EDTA.
Sample_label_protocol_ch1	Cleanup of Double-Stranded cDNA
Sample_label_protocol_ch1	Phenol/Chloroform Extraction
Sample_label_protocol_ch1	1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
Sample_label_protocol_ch1	2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
Sample_label_protocol_ch1	Ethanol Precipitation
Sample_label_protocol_ch1	1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
Sample_label_protocol_ch1	2.Immediately spin at maximum speed for 20 minutes at room temperature.
Sample_label_protocol_ch1	3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	5.Air dry pellet.
Sample_label_protocol_ch1	6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
Sample_label_protocol_ch1	Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
Sample_label_protocol_ch1	IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
Sample_label_protocol_ch1	1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
Sample_label_protocol_ch1	2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
Sample_label_protocol_ch1	IVT Cleanup
Sample_label_protocol_ch1	-The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
Sample_label_protocol_ch1	-The RNA elution step is performed twice to increase recovery.
Sample_label_protocol_ch1	-Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
Sample_label_protocol_ch1	Quantifying the cRNA
Sample_label_protocol_ch1	1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
Sample_label_protocol_ch1	2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
Sample_label_protocol_ch1	3.The following formula is used to determine the cRNA yield.
Sample_label_protocol_ch1	Adjusted cRNA yield=RNA* - (total RNA#)($)
Sample_label_protocol_ch1 = RNA*	amount of cRNA measured after IVT (mg)
Sample_label_protocol_ch1 = total RNA#	sterting amount of total RNA (mg)
Sample_label_protocol_ch1 = $	fraction of cRNA reaction used in IVT
Sample_label_protocol_ch1	cRNA Fragmentation
Sample_label_protocol_ch1	1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
Sample_label_protocol_ch1	2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
Sample_label_protocol_ch1	3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
Sample_label_protocol_ch1	Gel Electrophoresis
Sample_label_protocol_ch1	1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
Sample_label_protocol_ch1	2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
Sample_hyb_protocol	1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
Sample_hyb_protocol	2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
Sample_hyb_protocol	3.Prepare the hybridization cocktail as follows:
Sample_hyb_protocol	Reagents Mini Array Midi Array Standard Array
Sample_hyb_protocol	Fragmented cRNA 5mg 10mg 15 mg
Sample_hyb_protocol	Control oligo B2 1.7ml 3.3ml 5ml
Sample_hyb_protocol	20X eukaryotic hybridization controls 5ml 10ml 15ml
Sample_hyb_protocol	Herring sperm DNA 1ml 2ml 3ml
Sample_hyb_protocol	Acetylated BSA 1ml 2ml 3ml
Sample_hyb_protocol	2X Hybridization buffer 50ml 100ml 150mul
Sample_hyb_protocol	Water to 100ml to 200ml to 300ml
Sample_hyb_protocol	4.Equilibrate probe array to room temperature.
Sample_hyb_protocol	5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
Sample_hyb_protocol	6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
Sample_hyb_protocol	7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
Sample_hyb_protocol	8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
Sample_hyb_protocol	Washing and Staining Probe Arrays
Sample_hyb_protocol	Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
Sample_scan_protocol	The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
Sample_data_processing	dChip (version 1.3) scaled. Normalized.
Sample_platform_id	GPL1261
Sample_contact_name	Rui,,Zhu
Sample_contact_email	rzhu@mdanderson.org
Sample_contact_laboratory	Matin
Sample_contact_department	Genetics
Sample_contact_institute	UT MD Anderson Cancer Center
Sample_contact_address	1515 Holcombe Blvd.
Sample_contact_city	Houston
Sample_contact_state	TX
Sample_contact_zip/postal_code	77030
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM358nnn/GSM358306/suppl/GSM358306.CEL.gz
Sample_series_id	GSE14354
Sample_data_row_count	45101

GSM358307

GPL1261

PN1 Mouse testis_129/Sv

postnatal day 1 Mouse testis 129/Sv

Background Srain: 129/Sv Gender: male Tissue: testis Age: postnatal day 1

gene expression differences between 129/Sv and 129.MOLF-Chr19 strains

Sample_geo_accession	GSM358307
Sample_status	Public on Jan 13 2009
Sample_submission_date	Jan 07 2009
Sample_last_update_date	Jan 12 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	12 pairs of PN1 testes were pooled. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
Sample_label_protocol_ch1	Use a starting amount of 5.0 – 40.0 mg total RNA.
Sample_label_protocol_ch1	First Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
Sample_label_protocol_ch1	2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
Sample_label_protocol_ch1	4.Incubate at 42° C for 2 minutes.
Sample_label_protocol_ch1	5.Add to the tube 1 mL of Superscript II reverse transcriptase.
Sample_label_protocol_ch1	6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
Sample_label_protocol_ch1	2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
Sample_label_protocol_ch1	3.Add 2 mL [10 U] T4 DNA Polymerase.
Sample_label_protocol_ch1	4.Incubate at 16° C for 5 minutes.
Sample_label_protocol_ch1	5.Add 10 mL 0.5 M EDTA.
Sample_label_protocol_ch1	Cleanup of Double-Stranded cDNA
Sample_label_protocol_ch1	Phenol/Chloroform Extraction
Sample_label_protocol_ch1	1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
Sample_label_protocol_ch1	2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
Sample_label_protocol_ch1	Ethanol Precipitation
Sample_label_protocol_ch1	1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
Sample_label_protocol_ch1	2.Immediately spin at maximum speed for 20 minutes at room temperature.
Sample_label_protocol_ch1	3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	5.Air dry pellet.
Sample_label_protocol_ch1	6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
Sample_label_protocol_ch1	Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
Sample_label_protocol_ch1	IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
Sample_label_protocol_ch1	1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
Sample_label_protocol_ch1	2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
Sample_label_protocol_ch1	IVT Cleanup
Sample_label_protocol_ch1	-The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
Sample_label_protocol_ch1	-The RNA elution step is performed twice to increase recovery.
Sample_label_protocol_ch1	-Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
Sample_label_protocol_ch1	Quantifying the cRNA
Sample_label_protocol_ch1	1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
Sample_label_protocol_ch1	2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
Sample_label_protocol_ch1	3.The following formula is used to determine the cRNA yield.
Sample_label_protocol_ch1	Adjusted cRNA yield=RNA* - (total RNA#)($)
Sample_label_protocol_ch1 = RNA*	amount of cRNA measured after IVT (mg)
Sample_label_protocol_ch1 = total RNA#	sterting amount of total RNA (mg)
Sample_label_protocol_ch1 = $	fraction of cRNA reaction used in IVT
Sample_label_protocol_ch1	cRNA Fragmentation
Sample_label_protocol_ch1	1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
Sample_label_protocol_ch1	2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
Sample_label_protocol_ch1	3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
Sample_label_protocol_ch1	Gel Electrophoresis
Sample_label_protocol_ch1	1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
Sample_label_protocol_ch1	2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
Sample_hyb_protocol	1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
Sample_hyb_protocol	2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
Sample_hyb_protocol	3.Prepare the hybridization cocktail as follows:
Sample_hyb_protocol	Reagents Mini Array Midi Array Standard Array
Sample_hyb_protocol	Fragmented cRNA 5mg 10mg 15 mg
Sample_hyb_protocol	Control oligo B2 1.7ml 3.3ml 5ml
Sample_hyb_protocol	20X eukaryotic hybridization controls 5ml 10ml 15ml
Sample_hyb_protocol	Herring sperm DNA 1ml 2ml 3ml
Sample_hyb_protocol	Acetylated BSA 1ml 2ml 3ml
Sample_hyb_protocol	2X Hybridization buffer 50ml 100ml 150mul
Sample_hyb_protocol	Water to 100ml to 200ml to 300ml
Sample_hyb_protocol	4.Equilibrate probe array to room temperature.
Sample_hyb_protocol	5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
Sample_hyb_protocol	6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
Sample_hyb_protocol	7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
Sample_hyb_protocol	8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
Sample_hyb_protocol	Washing and Staining Probe Arrays
Sample_hyb_protocol	Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
Sample_scan_protocol	The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
Sample_data_processing	dChip (version 1.3) scaled. Normalized.
Sample_platform_id	GPL1261
Sample_contact_name	Rui,,Zhu
Sample_contact_email	rzhu@mdanderson.org
Sample_contact_laboratory	Matin
Sample_contact_department	Genetics
Sample_contact_institute	UT MD Anderson Cancer Center
Sample_contact_address	1515 Holcombe Blvd.
Sample_contact_city	Houston
Sample_contact_state	TX
Sample_contact_zip/postal_code	77030
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM358nnn/GSM358307/suppl/GSM358307.CEL.gz
Sample_series_id	GSE14354
Sample_data_row_count	45101

GSM358308

GPL1261

PN1 Mouse testis_M19

postnatal day 1 Mouse testis 129.MOLF-Chr19

Background Srain: 129.MOLF-Chr 19 Gender: male Tissue: testis Age: postnatal day 1

gene expression differences between 129/Sv and 129.MOLF-Chr19 strains

Sample_geo_accession	GSM358308
Sample_status	Public on Jan 13 2009
Sample_submission_date	Jan 07 2009
Sample_last_update_date	Jan 12 2009
Sample_type	RNA
Sample_channel_count	1
Sample_organism_ch1	Mus musculus
Sample_taxid_ch1	10090
Sample_molecule_ch1	total RNA
Sample_extract_protocol_ch1	25 pairs of PN1 testes were pooled. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
Sample_label_ch1	Biotin
Sample_label_protocol_ch1	Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
Sample_label_protocol_ch1	Use a starting amount of 5.0 – 40.0 mg total RNA.
Sample_label_protocol_ch1	First Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
Sample_label_protocol_ch1	2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
Sample_label_protocol_ch1	4.Incubate at 42° C for 2 minutes.
Sample_label_protocol_ch1	5.Add to the tube 1 mL of Superscript II reverse transcriptase.
Sample_label_protocol_ch1	6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
Sample_label_protocol_ch1	Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
Sample_label_protocol_ch1	1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
Sample_label_protocol_ch1	2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
Sample_label_protocol_ch1	3.Add 2 mL [10 U] T4 DNA Polymerase.
Sample_label_protocol_ch1	4.Incubate at 16° C for 5 minutes.
Sample_label_protocol_ch1	5.Add 10 mL 0.5 M EDTA.
Sample_label_protocol_ch1	Cleanup of Double-Stranded cDNA
Sample_label_protocol_ch1	Phenol/Chloroform Extraction
Sample_label_protocol_ch1	1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
Sample_label_protocol_ch1	2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
Sample_label_protocol_ch1	Ethanol Precipitation
Sample_label_protocol_ch1	1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
Sample_label_protocol_ch1	2.Immediately spin at maximum speed for 20 minutes at room temperature.
Sample_label_protocol_ch1	3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
Sample_label_protocol_ch1	5.Air dry pellet.
Sample_label_protocol_ch1	6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
Sample_label_protocol_ch1	Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
Sample_label_protocol_ch1	IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
Sample_label_protocol_ch1	1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
Sample_label_protocol_ch1	2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
Sample_label_protocol_ch1	IVT Cleanup
Sample_label_protocol_ch1	-The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
Sample_label_protocol_ch1	-The RNA elution step is performed twice to increase recovery.
Sample_label_protocol_ch1	-Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
Sample_label_protocol_ch1	Quantifying the cRNA
Sample_label_protocol_ch1	1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
Sample_label_protocol_ch1	2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
Sample_label_protocol_ch1	3.The following formula is used to determine the cRNA yield.
Sample_label_protocol_ch1	Adjusted cRNA yield=RNA* - (total RNA#)($)
Sample_label_protocol_ch1 = RNA*	amount of cRNA measured after IVT (mg)
Sample_label_protocol_ch1 = total RNA#	sterting amount of total RNA (mg)
Sample_label_protocol_ch1 = $	fraction of cRNA reaction used in IVT
Sample_label_protocol_ch1	cRNA Fragmentation
Sample_label_protocol_ch1	1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
Sample_label_protocol_ch1	2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
Sample_label_protocol_ch1	3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
Sample_label_protocol_ch1	Gel Electrophoresis
Sample_label_protocol_ch1	1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
Sample_label_protocol_ch1	2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
Sample_hyb_protocol	1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
Sample_hyb_protocol	2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
Sample_hyb_protocol	3.Prepare the hybridization cocktail as follows:
Sample_hyb_protocol	Reagents Mini Array Midi Array Standard Array
Sample_hyb_protocol	Fragmented cRNA 5mg 10mg 15 mg
Sample_hyb_protocol	Control oligo B2 1.7ml 3.3ml 5ml
Sample_hyb_protocol	20X eukaryotic hybridization controls 5ml 10ml 15ml
Sample_hyb_protocol	Herring sperm DNA 1ml 2ml 3ml
Sample_hyb_protocol	Acetylated BSA 1ml 2ml 3ml
Sample_hyb_protocol	2X Hybridization buffer 50ml 100ml 150mul
Sample_hyb_protocol	Water to 100ml to 200ml to 300ml
Sample_hyb_protocol	4.Equilibrate probe array to room temperature.
Sample_hyb_protocol	5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
Sample_hyb_protocol	6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
Sample_hyb_protocol	7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
Sample_hyb_protocol	8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
Sample_hyb_protocol	Washing and Staining Probe Arrays
Sample_hyb_protocol	Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
Sample_scan_protocol	The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
Sample_data_processing	dChip (version 1.3) scaled. Normalized.
Sample_platform_id	GPL1261
Sample_contact_name	Rui,,Zhu
Sample_contact_email	rzhu@mdanderson.org
Sample_contact_laboratory	Matin
Sample_contact_department	Genetics
Sample_contact_institute	UT MD Anderson Cancer Center
Sample_contact_address	1515 Holcombe Blvd.
Sample_contact_city	Houston
Sample_contact_state	TX
Sample_contact_zip/postal_code	77030
Sample_contact_country	USA
Sample_supplementary_file	ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM358nnn/GSM358308/suppl/GSM358308.CEL.gz
Sample_series_id	GSE14354
Sample_data_row_count	45101

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Transcripts profile based on;

A. Differential status (Up/Down regulation)

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