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Sample GSM359513 Query DataSets for GSM359513
Status Public on Aug 14, 2009
Title Spurp_CO2_Hyb9_B2O2
Sample type RNA
 
Channel 1
Source name S. purpuratus, prism, 1020ppm CO2, rep2
Organism Strongylocentrotus purpuratus
Characteristics Species: Strongylocentrotus purpuratus,
Developmental stage: prism,
Age: 40 hours post-fertilization,
CO2 treatment: 1020 ppm CO2,
Replicate culture: 2
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from a pooled sample of approximate 60,000 prism stage urchin larvae. Total RNA was isolated using TRIzol® Reagent following manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). RNA was then processed through an additional column clean-up step to remove tRNA and small-sized RNA degradation products.
Label Alexafluor 647
Label protocol Indirect coupling of dye following cDNA synthesis with amino-allyl dUTP. cDNA was incubated with 1 μL of either Alexafluor 555 or 647 dye for 1h at room temperature in the dark.
 
Channel 2
Source name S. purpuratus, prism, 380ppm CO2, rep2
Organism Strongylocentrotus purpuratus
Characteristics Species: Strongylocentrotus purpuratus,
Developmental stage: prism,
Age: 40 hours post-fertilization,
CO2 treatment: 380 ppm CO2,
Replicate culture: 2
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from a pooled sample of approximate 60,000 prism stage urchin larvae. Total RNA was isolated using TRIzol® Reagent following manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). RNA was then processed through an additional column clean-up step to remove tRNA and small-sized RNA degradation products.
Label Alexafluor 555
Label protocol Indirect coupling of dye following cDNA synthesis with amino-allyl dUTP. cDNA was incubated with 1 μL of either Alexafluor 555 or 647 dye for 1h at room temperature in the dark.
 
 
Hybridization protocol Samples were hybridized following Agilent’s Protocol for Two-Color Microarray-Based Gene Expression Analysis (version 5.5, pages 24-35). The cDNA samples were quantified using a NanoDrop 1000 UV/visible spectrophotometer (Thermo Scientific, Wilmington, DE, USA) to ensure high quality cDNA synthesis and dye incorporation before continuing on to the slide hybridization. If the cDNA yield of a particular sample was <600 ηg and the specific activity was <8.0 ρmol dye per μg cDNA, cDNA synthesis and labeling was repeated. Prior to slide hybridization, cDNA samples were fragmented following Agilent’s Gene Expression Hybrization Kit for 4x44K slides. Briefly, 600ηg of Alexafluor 555-labeled cDNA, 600ηg of Alexafluor 647-labeled cDNA, 11 μL of 10X Blocking Agent and 2.2 μL of 25X Fragmentation Buffer were mixed to a total volume of 55 μL with nuclease-free water and incubated at 60°C for 30 min. To each array mixture 55 μL of 2X GE Hybridization Buffer HI-RPM was added and then the sample was applied to the microarray slides. Hybridizations were conducted overnight (~17 h) at 65°C in Agilent Microarray Hybridization Chambers with the hybridization oven rotisserie set at 10rpm. Following hybridization, the slides were washed in Agilent’s Gene Expression Wash Buffers following manufacturer’s instructions.
Scan protocol The slides were scanned at 5μm on a GenePix 4000B microarray scanner (Axon Instruments, Molecular Devices, Sunnyvale, CA, USA). Data from the microarrays were extracted using GenePix Pro 4.0 software (Axon Instruments).
Description N/A
Data processing Normalization and data analysis were completed using R (R Development Core Team 2008) with the limma software package (Smyth 2005). To avoid violating assumptions of the number or degree of symmetry of differentially expressed genes, a global normalization for dye-bias was applied on a probe-by-probe basis, after averaging over log-ratios from replicate probes within arrays, by including a dye-effect in the linear model.
 
Submission date Jan 12, 2009
Last update date Aug 14, 2009
Contact name Anne Todgham
E-mail(s) [email protected]
Organization name San Francisco State University
Department Biology
Street address 1600 Holloway Ave
City San Francisco
State/province CA
ZIP/Postal code 94132-1722
Country USA
 
Platform ID GPL7011
Series (1)
GSE13777 Purple sea urchin larvae alter their transcriptome in response to ocean acidification

Data table header descriptions
ID_REF
Probe ID Probe identification for each GLEAN3
VALUE Normalized Log Ratio (F635/F532)
CH1_SIG_MEAN Normalized Mean F635 Intensity
CH2_SIG_MEAN Normalized Mean F532 Intensity

Data table
ID_REF Probe ID VALUE CH1_SIG_MEAN CH2_SIG_MEAN
1 >GLEAN3_00022_1 -0.183131834 1345.668984 1527.79969
2 >GLEAN3_00022_2 -0.113642339 2254.878933 2439.68065
3 >GLEAN3_00022_3 -0.112885314 1838.843272 1988.504511
4 >GLEAN3_00046_1 -0.042744179 243.7283438 251.057568
5 >GLEAN3_00046_2 -0.048194842 519.86898 537.529121
6 >GLEAN3_00046_3 -0.118629083 201.3375971 218.59277
7 >GLEAN3_00048_1 -0.278892025 834.9147796 1012.971004
8 >GLEAN3_00048_2 -0.232243479 906.5958637 1064.942077
9 >GLEAN3_00048_3 -0.393067934 835.0965323 1096.634536
10 >GLEAN3_00073_1 -0.100952532 165.3453315 177.3297809
11 >GLEAN3_00073_2 -0.218962798 168.638499 196.2777624
12 >GLEAN3_00073_3 0.17214359 695.9078963 617.6342627
13 >GLEAN3_00129_1 -0.110256553 266.5793237 287.7510993
14 >GLEAN3_00129_2 -0.229827289 273.7963296 321.0794643
15 >GLEAN3_00129_3 -0.187438565 239.6771617 272.9300052
16 >GLEAN3_00142_1 -0.218380473 703.1856443 818.1050411
17 >GLEAN3_00142_2 -0.42214701 5286.892337 7084.013485
18 >GLEAN3_00142_3 -0.109372826 745.3989518 804.1059562
19 >GLEAN3_00151_1 0.122648481 318.880076 292.8913281
20 >GLEAN3_00151_2 0.232601913 356.1579426 303.1255062

Total number of rows: 2986

Table truncated, full table size 166 Kbytes.




Supplementary file Size Download File type/resource
GSM359513.gpr.gz 3.5 Mb (ftp)(http) GPR
Processed data included within Sample table

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