Comment[ArrayExpressAccession] E-GEOD-43982 MAGE-TAB Version 1.1 Public Release Date 2013-05-01 Investigation Title Transcriptome analysis of multiple pre-meiotic anther stages and laser microdissected cell types in maize Comment[Submitted Name] Transcriptome analysis of multiple pre-meiotic anther stages and laser microdissected cell types in maize Experiment Description Transcriptomes from multiple pre-meiotic stages of wild type, mac1, and msca1 maize anthers were characterized by microarray hybridization. The goal was to characterize the developmental progression as the anther specifies five cell types and grows rapidly precedeing meiotic entry. The stages characterized were immature anther primordia (0.15 mm long in maize) containing just stem cells, through somatic and germinal cell fate specification (0.20 and 0.25 mm), mitotic proliferation (0.4 mm), and finally the birth of the middle layer and tapetum (0.7 mm). To obtain cell-type specific markers, at 0.7 mm we also compared whole anthers to collections of laser-microdissected anther cell types including the archesporial cells (pre-meiotic germinal cells), nutritive layers (middle layer and tapetum) and structural layers (endothecium and epidemis) of the anther lobe. keyword: anther development, maize, male-sterile Three loop designs covered the early stages (up to 0.7 mm) with two replicates for each comparison. The first loop had 0.2 mm long anthers and compared wild type versus mac1 mutant versus msca1 mutant in a three vertex loop design. The second loop had four vertices and compared 0.15 mm WT anther primordia, 0.25 mm WT anthers, 0.4 mm WT anthers and finally 0.4 mm mac1 mutant anthers. The third had 0.7 mm anthers in a three vertex loop with the nutritive layers (middle layer and tapetum) at one vertex, the germinal pre-meiotic cells at another vertex, and whole anthers at a third vertex. The whole anther samples were also, separately and outside of the loop, compared in four replicates to the structural layers (endothecium and epidermis). Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Fernandes Timothy Walbot Person First Name John Kelliher Virginia Person Mid Initials F Person Email jfernand@stanford.edu Person Affiliation Stanford University Person Phone 650-533-9376 Person Address Bio Sci, Stanford University, 385 Serra Mall, Stanford, CA, USA Person Roles submitter Protocol Name P-GSE43982-1 P-GSE43982-2 P-GSE43982-6 P-GSE43982-7 P-GSE43982-3 P-GSE43982-4 P-GSE43982-5 P-GSE43982-8 Protocol Description After removal of spots flagged by Feature Extraction as below background or saturated or outliers, intensities for each channel from Feature Extraction were normalized using lowess. Average intensities were then calculated for each sample type for comparison. The limma package of R was used to normalize the median intensities for each channel as follows: within each array using loess and between arrays using quantile method. ID_REF = VALUE = normalized After removal of spots flagged by Feature Extraction as below background or saturated or outliers, intensities for each channel from Feature Extraction were normalized using lowess. Average intensities were then calculated for each sample type for comparison. The limma package of R was used to normalize the median intensities for each channel as follows: within each array using loess and between arrays using method. ID_REF = VALUE = normalized Total RNA (50 ng) was amplified & labeled with Cy3 or Cy5 using Agilent Low RNA Input Linear Amplification Kit. 825 ng of each labeled target cRNA were hybridized for 17 h at 60C. None Plants were grown and dissected at the Stanford field; tissues were collected and frozen in liquid nitrogen immediately. For whole anthers, total RNA was extracted from 30 - 60 mg of frozen tissues with TRIzol, digested with DNase I, and cleaned up with Qiagen RNeasy Mini kit. For laser microdissection, a PALM microbeam LCM was used for dissection, and RNA was extracted and cleaned up with a PicoPure column (Arcturus), treated with Dnase I, and resuspended in dH2O. Slides were scanned on an Agilent DNA microarray scanner as indicated in the manual. Images were processed with the Agilent Feature Extraction software. Protocol Type normalization data transformation protocol normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name ORGANISM PART Experimental Factor Type organism part Comment[SecondaryAccession] GSE43982 Comment[GEOReleaseDate] 2013-05-01 Comment[ArrayExpressSubmissionDate] 2013-02-01 Comment[GEOLastUpdateDate] 2013-05-01 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE43982_US84703594_251604710097_S01_GE2-v5_95_Feb07_2_JF_1_1.txt Comment[AdditionalFile:Data2] GSE43982_US84703594_251604710097_S01_GE2-v5_95_Feb07_2_JF_1_2.txt Comment[AdditionalFile:Data3] GSE43982_US84703594_251604710097_S01_GE2-v5_95_Feb07_2_JF_1_3.txt Comment[AdditionalFile:Data4] GSE43982_US84703594_251604710097_S01_GE2-v5_95_Feb07_2_JF_1_4.txt Comment[AdditionalFile:Data5] GSE43982_US84703594_251604710103_S01_GE2-v5_95_Feb07_2_JF_1_1.txt Comment[AdditionalFile:Data6] GSE43982_US84703594_251604710103_S01_GE2-v5_95_Feb07_2_JF_1_2.txt Comment[AdditionalFile:Data7] GSE43982_US84703594_251604710103_S01_GE2-v5_95_Feb07_2_JF_1_3.txt Comment[AdditionalFile:Data8] GSE43982_US84703594_251604710103_S01_GE2-v5_95_Feb07_2_JF_1_4.txt Comment[AdditionalFile:Data9] GSE43982_US84703594_251604710247_S01_GE2-v5_95_Feb07_2_JF_1_1.txt Comment[AdditionalFile:Data10] GSE43982_US84703594_251604710247_S01_GE2-v5_95_Feb07_2_JF_1_2.txt Comment[AdditionalFile:Data11] GSE43982_US84703594_251604710247_S01_GE2-v5_95_Feb07_2_JF_1_3.txt Comment[AdditionalFile:Data12] GSE43982_US84703594_251604710247_S01_GE2-v5_95_Feb07_2_JF_1_4.txt Comment[AdditionalFile:Data13] GSE43982_US84703594_251604710248_S01_GE2-v5_95_Feb07_2_JF_1_1.txt Comment[AdditionalFile:Data14] GSE43982_US84703594_251604710248_S01_GE2-v5_95_Feb07_2_JF_1_2.txt Comment[AdditionalFile:Data15] GSE43982_US84703594_251604710248_S01_GE2-v5_95_Feb07_2_JF_1_3.txt Comment[AdditionalFile:Data16] GSE43982_US84703594_251604710248_S01_GE2-v5_95_Feb07_2_JF_1_4.txt Comment[AdditionalFile:Data17] GSE43982_US84703594_251604710253_S01_GE2-v5_95_Feb07_2_JF_1_1.txt Comment[AdditionalFile:Data18] GSE43982_US84703594_251604710253_S01_GE2-v5_95_Feb07_2_JF_1_2.txt Comment[AdditionalFile:Data19] GSE43982_US84703594_251604710253_S01_GE2-v5_95_Feb07_2_JF_1_3.txt Comment[AdditionalFile:Data20] GSE43982_US84703594_251604710253_S01_GE2-v5_95_Feb07_2_JF_1_4.txt Comment[AdditionalFile:Data21] GSE43982_US84703594_251604710254_S01_GE2-v5_95_Feb07_2_JF_1_1.txt Comment[AdditionalFile:Data22] GSE43982_US84703594_251604710254_S01_GE2-v5_95_Feb07_2_JF_1_2.txt Comment[AdditionalFile:Data23] GSE43982_US84703594_251604710254_S01_GE2-v5_95_Feb07_2_JF_1_3.txt Comment[AdditionalFile:Data24] GSE43982_US84703594_251604710254_S01_GE2-v5_95_Feb07_2_JF_1_4.txt SDRF File E-GEOD-43982.sdrf.txt