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^SAMPLE = Control Embyronic Stem Cell Replicate 1
!Sample_title = Control Embyronic Stem Cell Replicate 1
!Sample_supplementary_file = file1.gpr
!Sample_source_name_ch1 = Total RNA from murine ES-D3 embryonic stem cells labeled with Cyanine-5 (red).
!Sample_organism_ch1 = Mus musculus
!Sample_characteristics_ch1 = ES-D3 cell line (CRL-1934)
!Sample_characteristics_ch1 = Age: day 4
!Sample_characteristics_ch1 = Tissue: blastocytes
!Sample_characteristics_ch1 = Strain: 129/Sv mice
!Sample_growth_protocol_ch1 = ES cells were kept in an undifferentiated, pluripotent state by using 1000 IU/ml leukemia inhibitory factor (LIF; Chemicon, ESGRO, ESG1107), and grown on top of murine embryonic fibroblasts feeder layer inactivated by 10 ug/ml of mitomycin C (Sigma, St. Louis). ES cells were cultured on 0.1% gelatin-coated plastic dishes in ES medium containing Dulbecco modified Eagle medium supplemented with 15% fetal calf serum, 0.1 mM beta-mercaptoethanol, 2 mM glutamine, and 0.1 mN non-essential amino acids.
!Sample_extract_protocol_ch1 = TriZol procedure
!Sample_molecule_ch1 = total RNA
!Sample_label_ch1 = Cy5
!Sample_label_protocol_ch1 = 10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy5-labeled dCTP (NEN Life Science, Boston, MA), and RNase inhibitor (Invitrogen). RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns (Qiagen).
!Sample_source_name_ch2 = Total RNA from pooled whole mouse embryos e17.5, labeled with Cyanine-3 (green).
!Sample_organism_ch2 = Mus musculus
!Sample_characteristics_ch2 = Strain: C57BL/6
!Sample_characteristics_ch2 = Age: e17.5 d
!Sample_characteristics_ch2 = Tissue: whole embryo
!Sample_extract_protocol_ch2 = TriZol procedure
!Sample_molecule_ch2 = total RNA
!Sample_label_ch2 = Cy3
!Sample_label_protocol_ch2 = 10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy5-labeled dCTP (NEN Life Science, Boston, MA), and RNase inhibitor (Invitrogen). RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns (Qiagen).
!Sample_hyb_protocol = Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially with 6x SSC/0.005% Triton X-102 and 0.1x SSC/0.005% Triton X-102 before scanning. Slides were hybridized for 17 h at 60°C in a rotating oven, and washed.
!Sample_scan_protocol = Scanned on an Agilent G2565AA scanner.
!Sample_scan_protocol = Images were quantified using Agilent Feature Extraction Software (version A.7.5).
!Sample_description = Biological replicate 1 of 4. Control embryonic stem cells, untreated, harvested after several passages.
!Sample_data_processing = LOWESS normalized, background subtracted VALUE data obtained from log of processed Red signal/processed Green signal.
!Sample_platform_id = GPL3759
#ID_REF =
#VALUE = log(REDsignal/GREENsignal) per feature (processed signals used).
#LogRatioError = error of the log ratio calculated according to the error model chosen.
#PValueLogRatio = Significance level of the Log Ratio computed for a feature.
#gProcessedSignal = Dye-normalized signal after surrogate "algorithm," green "channel," used for computation of log ratio.
#rProcessedSignal = Dye-normalized signal after surrogate "algorithm," red "channel," used for computation of log ratio.
!sample_table_begin
ID_REF VALUE LogRatioError PValueLogRatio gProcessedSignal rProcessedSignal
1 -1.6274758 1.36E-01 6.41E-33 9.13E+03 2.15E+02
2 0.1412248 1.34E+00 1.00E+00 4.14E+01 5.72E+01
3 0.1827684 5.19E-02 4.33E-04 5.13E+03 7.81E+03
4 -0.3932267 6.08E-02 1.02E-10 4.65E+03 1.88E+03
5 -0.9865994 1.05E-01 6.32E-21 2.91E+03 3.01E+02
6 0.0238812 1.02E-01 8.15E-01 7.08E+02 7.48E+02
7 -1.4841822 1.25E-01 1.42E-32 1.02E+04 3.36E+02
8 -1.8261356 4.15E-01 1.10E-05 7.19E+02 1.07E+01
9 -1.0344779 1.78E+00 1.00E+00 9.62E+01 8.89E+00
10 0.2405891 3.09E-01 4.36E-01 1.61E+02 2.80E+02
11 0.3209366 3.59E-01 3.71E-01 1.25E+02 2.61E+02
12 0.358304 2.06E+00 1.00E+00 2.04E+01 4.66E+01
13 -0.0122072 3.64E-01 9.73E-01 1.84E+02 1.79E+02
14 -1.5480396 1.30E-01 7.21E-33 1.02E+04 2.90E+02
15 0.0073419 2.98E-01 9.80E-01 2.21E+02 2.25E+02
16 -0.2267015 9.44E-01 8.10E-01 8.90E+01 5.28E+01
17 -0.1484023 8.01E-01 8.53E-01 9.65E+01 6.86E+01
18 -0.6122195 1.28E-01 1.69E-06 1.12E+03 2.73E+02
19 0.0796905 8.78E-02 3.64E-01 8.21E+02 9.87E+02
20 -0.084895 9.38E-01 9.28E-01 7.68E+01 6.32E+01
!sample_table_end
^SAMPLE = Control Embyronic Stem Cell Replicate 2
!Sample_title = Control Embyronic Stem Cell Replicate 2
!Sample_supplementary_file = file2.gpr
!Sample_source_name_ch1 = Total RNA from murine ES-D3 embryonic stem cells labeled with Cyanine-5 (red).
!Sample_organism_ch1 = Mus musculus
!Sample_characteristics_ch1 = ES-D3 cell line (CRL-1934)
!Sample_characteristics_ch1 = Age: day 4
!Sample_characteristics_ch1 = Tissue: blastocytes
!Sample_characteristics_ch1 = Strain: 129/Sv mice
!Sample_growth_protocol_ch1 = ES cells were kept in an undifferentiated, pluripotent state by using 1000 IU/ml leukemia inhibitory factor (LIF; Chemicon, ESGRO, ESG1107), and grown on top of murine embryonic fibroblasts feeder layer inactivated by 10 ug/ml of mitomycin C (Sigma, St. Louis). ES cells were cultured on 0.1% gelatin-coated plastic dishes in ES medium containing Dulbecco modified Eagle medium supplemented with 15% fetal calf serum, 0.1 mM beta-mercaptoethanol, 2 mM glutamine, and 0.1 mN non-essential amino acids.
!Sample_extract_protocol_ch1 = TriZol procedure
!Sample_molecule_ch1 = total RNA
!Sample_label_ch1 = Cy5
!Sample_label_protocol_ch1 = 10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy5-labeled dCTP (NEN Life Science, Boston, MA), and RNase inhibitor (Invitrogen). RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns (Qiagen).
!Sample_source_name_ch2 = Total RNA from pooled whole mouse embryos e17.5, labeled with Cyanine-3 (green).
!Sample_organism_ch2 = Mus musculus
!Sample_characteristics_ch2 = Strain: C57BL/6
!Sample_characteristics_ch2 = Age: e17.5 d
!Sample_characteristics_ch2 = Tissue: whole embryo
!Sample_extract_protocol_ch2 = TriZol procedure
!Sample_molecule_ch2 = total RNA
!Sample_label_ch2 = Cy3
!Sample_label_protocol_ch2 = 10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy5-labeled dCTP (NEN Life Science, Boston, MA), and RNase inhibitor (Invitrogen). RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns (Qiagen).
!Sample_hyb_protocol = Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially with 6x SSC/0.005% Triton X-102 and 0.1x SSC/0.005% Triton X-102 before scanning. Slides were hybridized for 17 h at 60°C in a rotating oven, and washed.
!Sample_scan_protocol = Scanned on an Agilent G2565AA scanner.
!Sample_scan_protocol = Images were quantified using Agilent Feature Extraction Software (version A.7.5).
!Sample_description = Biological replicate 2 of 4. Control embryonic stem cells, untreated, harvested after several passages.
!Sample_data_processing = LOWESS normalized, background subtracted VALUE data obtained from log of processed Red signal/processed Green signal.
!Sample_platform_id = GPL3759
#ID_REF =
#VALUE = log(REDsignal/GREENsignal) per feature (processed signals used).
#LogRatioError = error of the log ratio calculated according to the error model chosen.
#PValueLogRatio = Significance level of the Log Ratio computed for a feature.
#gProcessedSignal = Dye-normalized signal after surrogate "algorithm," green "channel," used for computation of log ratio.
#rProcessedSignal = Dye-normalized signal after surrogate "algorithm," red "channel," used for computation of log ratio.
!sample_table_begin
ID_REF VALUE LogRatioError PValueLogRatio gProcessedSignal rProcessedSignal
1 -1.1697263 1.23E-01 2.14E-21 3.17E+03 2.14E+02
2 -0.1111353 1.63E+00 9.46E-01 5.43E+01 4.20E+01
3 0.1400597 5.11E-02 6.17E-03 6.72E+03 9.28E+03
4 -0.4820633 6.38E-02 4.06E-14 6.46E+03 2.13E+03
5 -1.2116196 1.22E-01 2.31E-23 3.62E+03 2.22E+02
6 -0.0230528 1.04E-01 8.24E-01 8.76E+02 8.31E+02
7 -1.1380152 1.13E-01 9.23E-24 3.94E+03 2.86E+02
8 -1.834596 5.40E-01 6.74E-04 6.44E+02 9.43E+00
9 -0.9747637 2.14E+00 1.00E+00 9.17E+01 9.72E+00
10 0.3874005 2.92E-01 1.85E-01 1.69E+02 4.11E+02
11 0.5340442 3.29E-01 1.04E-01 1.23E+02 4.20E+02
12 0.3260696 1.92E+00 8.65E-01 2.73E+01 5.77E+01
13 0.3010618 2.84E-01 2.90E-01 1.93E+02 3.87E+02
14 -1.0760413 1.08E-01 1.63E-23 4.06E+03 3.41E+02
15 -0.1167371 3.87E-01 7.63E-01 2.32E+02 1.77E+02
16 -0.1936322 9.44E-01 8.38E-01 1.02E+02 6.56E+01
17 -0.3275898 7.87E-01 6.77E-01 1.41E+02 6.65E+01
18 -0.4805853 1.14E-01 2.41E-05 1.34E+03 4.42E+02
19 0.1109524 9.56E-02 2.46E-01 8.38E+02 1.08E+03
20 0.1677912 6.51E-01 7.97E-01 9.84E+01 1.45E+02
!sample_table_end
^SAMPLE = Triple-Fusion Transfected Embryonic Stem Cells Replicate 1
!Sample_supplementary_file = file3.gpr
!Sample_title = Triple-Fusion Transfected Embryonic Stem Cells Replicate 1
!Sample_source_name_ch1 = Total RNA from murine ES-D3 triple-transfected embryonic stem cells labeled with Cyanine-5 (red).
!Sample_organism_ch1 = Mus musculus
!Sample_characteristics_ch1 = ES-D3 cell line (CRL-1934)
!Sample_characteristics_ch1 = Transfected with pUb-fluc-mrfp-ttk triple fusion reporter gene.
!Sample_characteristics_ch1 = Age: day 4
!Sample_characteristics_ch1 = Tissue: blastocytes
!Sample_characteristics_ch1 = Strain: 129/Sv mice
!Sample_treatment_protocol_ch1 = PCR amplification and standard cloning techniques were used to insert fluc and mrfp genes from plasmids pCDNA 3.1-CMV-fluc (Promega, Madison, WI) and pCDNA3.1-CMV-mrfp in frame with the ttk gene into the pCDNA3.1-truncated sr39tk. This triple fusion (TF) reporter gene fragment (3.3 kbp) was released from the plasmid with Not1 and BamH1 restriction enzymes before blunt-end ligation into the multiple cloning site of lentiviral transfer vector, FUG, driven by the human ubiquitin-C promoter. Self-inactivating (SIN) lentivirus was prepared by transient transfection of 293T cells. Briefly, pFUG-TF containing the triple fusion reporter gene was co-transfected into 293T cells with HIV-1 packaging vector (?8.9) and vesicular stomatitis virus G glycoprotein-pseudotyped envelop vector (pVSVG). Lentivirus supernatant was concentrated by sediment centrifugation using a SW29 rotor at 50,000 x g for two hours. Concentrated virus was titered on 293T cells. Murine ES cells were transfected with LV-pUb-fluc-mrfp-ttk at a multiplicity of infection (MOI) of 10.
!Sample_growth_protocol_ch1 = ES cells were kept in an undifferentiated, pluripotent state by using 1000 IU/ml leukemia inhibitory factor (LIF; Chemicon, ESGRO, ESG1107), and grown on top of murine embryonic fibroblasts feeder layer inactivated by 10 ug/ml of mitomycin C (Sigma, St. Louis). ES cells were cultured on 0.1% gelatin-coated plastic dishes in ES medium containing Dulbecco modified Eagle medium supplemented with 15% fetal calf serum, 0.1 mM beta-mercaptoethanol, 2 mM glutamine, and 0.1 mN non-essential amino acids.
!Sample_extract_protocol_ch1 = TriZol procedure
!Sample_molecule_ch1 = total RNA
!Sample_label_ch1 = Cy5
!Sample_label_protocol_ch1 = 10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP with 25 µM dCTP, 25 µM Cy5-labeled dCTP (NEN Life Science, Boston, MA), and RNase inhibitor (Invitrogen). RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns (Qiagen).
!Sample_source_name_ch2 = Total RNA from pooled whole mouse embryos e17.5, labeled with Cyanine-3 (green).
!Sample_organism_ch2 = Mus musculus
!Sample_characteristics_ch2 = Strain: C57BL/6
!Sample_characteristics_ch2 = Age: e17.5 d
!Sample_characteristics_ch2 = Tissue: whole embryo
!Sample_extract_protocol_ch2 = TriZol procedure
!Sample_molecule_ch2 = total RNA
!Sample_label_ch2 = Cy3
!Sample_label_protocol_ch2 = 10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy5-labeled dCTP (NEN Life Science, Boston, MA), and RNase inhibitor (Invitrogen). RNA was then degraded with RNase A, and labeled cDNAs were purified using QIAquick PCR columns (Qiagen).
!Sample_hyb_protocol = Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially with 6x SSC/0.005% Triton X-102 and 0.1x SSC/0.005% Triton X-102 before scanning. Slides were hybridized for 17 h at 60°C in a rotating oven, and washed.
!Sample_scan_protocol = Scanned on an Agilent G2565AA scanner.
!Sample_description = Biological replicate 1 of 3. Stable triple-fusion-reporter-gene transfected embryonic stem cells, harvested after several passages.
!Sample_data_processing = LOWESS normalized, background subtracted VALUE data obtained from log of processed Red signal/processed Green signal.
!Sample_platform_id = GPL3759
#ID_REF =
#VALUE = log(REDsignal/GREENsignal) per feature (processed signals used).
#LogRatioError = error of the log ratio calculated according to the error model chosen.
#PValueLogRatio = Significance level of the Log Ratio computed for a feature.
#gProcessedSignal = Dye-normalized signal after surrogate "algorithm," green "channel," used for computation of log ratio.
#rProcessedSignal = Dye-normalized signal after surrogate "algorithm," red "channel," used for computation of log ratio.
!sample_table_begin
ID_REF VALUE LogRatioError PValueLogRatio gProcessedSignal rProcessedSignal
1 -0.7837546 1.30E-01 1.70E-09 2.10E+03 3.46E+02
2 0.3797837 1.15E+00 7.41E-01 5.59E+01 1.34E+02
3 0.2079269 5.38E-02 1.12E-04 5.04E+03 8.14E+03
4 -0.4730291 6.71E-02 1.86E-12 5.66E+03 1.91E+03
5 -0.9481128 1.19E-01 1.30E-15 3.10E+03 3.49E+02
6 -0.0159867 1.33E-01 9.05E-01 8.45E+02 8.14E+02
7 -0.819922 1.14E-01 7.01E-13 2.75E+03 4.16E+02
8 -0.1559774 9.16E-01 8.65E-01 1.34E+02 9.34E+01
9 0.145267 3.90E+00 1.00E+00 2.22E+01 3.10E+01
10 0.3611211 3.40E-01 2.88E-01 1.97E+02 4.52E+02
11 0.5092089 4.39E-01 2.46E-01 1.24E+02 4.01E+02
12 0.3715387 1.69E+00 8.26E-01 3.84E+01 9.04E+01
13 0.1734934 3.57E-01 6.27E-01 2.37E+02 3.53E+02
14 -0.9340707 1.20E-01 6.90E-15 2.96E+03 3.45E+02
15 -0.2956317 5.78E-01 6.09E-01 2.46E+02 1.25E+02
16 -0.2321102 1.22E+00 8.49E-01 1.09E+02 6.37E+01
17 -0.1603561 1.16E+00 8.90E-01 1.06E+02 7.34E+01
18 -0.5063897 1.63E-01 1.95E-03 1.15E+03 3.58E+02
19 0.1990761 1.32E-01 1.32E-01 6.65E+02 1.05E+03
20 0.2985912 8.89E-01 7.37E-01 8.06E+01 1.60E+02
!sample_table_end
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