Computational protocol: Genome-Wide Transcript Profiling of Endosperm without Paternal Contribution Identifies Parent-of-Origin–Dependent Regulation of AGAMOUS-LIKE36

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Protocol publication

[…] Plants were grown and seeds isolated as described above. Total RNA was isolated as described above. For microarray analysis, three biological replicas were generated, each consisting of approximately 35 hand-pollinated siliques from ten different plants. The microarray experiment was conducted by the NARC Microarray Service in Trondheim. Microarray slides were printed by the Norwegian Microarray Consortium (Trondheim, Norway). A custom made Arabidopsis chip with 32567 unique 70-mer oligo probes was used in the experiments. Total RNA (15 mg) and Super-Script III reverse transcriptase (Invitrogen) were used in a reverse transcription reaction. A 3DNA Array 350 kit with Cy3- and Cy5-labelled dendrimers (Genisphere Inc.) was used for labeling. Hybridizations were performed in a Slide Booster Hybridization Station (Advalytix), and the slides were washed according to the manufacturers' descriptions (Genisphere and Advalytix). The slides were scanned at 10 mm resolution on a G2505B Agilent DNA microarray scanner (Agilent Technologies). The resulting image files were processed using GenePix 5.1 software (Axon Instruments). Spots identified as not found or manually flagged out as bad were filtered out. Spots with more than 50% saturated pixels were also excluded. The data sets were log-transformed and normalized using the print-tip Loess approach . Within-array replicated measurements for the same gene were merged by taking the average between the replicates. The data were then scaled so that all array data sets had the same median absolute deviation. The differentially expressed genes were identified using the Limma software package . The resulting set of p-values were used to compute the q-values as described .The microarray data generated in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE24809 ( [...] We defined the following sub-sets for our microarray data (see ): All expressed  =  all genes having a present call (17223 genes); Down 0.8 =  in Ler x cdka;1 downregulated genes with q≤0.35 and arithmetic ratio (ar) ≤0.8 (602 genes); Up 1.5 =  in Ler x cdka;1 upregulated genes with q≤0.35 and ar ≥1.5 (323 genes); Up 1.2 =  in Ler x cdka;1 upregulated genes with q≤0.35 and ar ≥1.2 (1030 genes). The q-value is the false discovery rate (FDR) of the p-value, and was adjusted with Storey's q-procedure . The threshold for analysis was set to q≤0.35 since this value detected paternally expressed genes at an arithmetic ratio (ar) ≤0.8. A functional classification was done at using the GO-Slim Molecular Function classification system. For the detailed transcription factor analysis we used the Transcription Factor (TF) classification from the Arabidopsis transcription factor database (AtTFDB) hosted on the Arabidopsis Gene Regulatory Information Server (AGRIS, The MADS TFs were sub-grouped as in de Folter et al . We compared our microarray data with seed expression data generated by the Goldberg & Harada laboratories, available at data comparison a reference set of genes was used that contained all genes covered by the Operon chip used in our study (Arabidopsis thaliana 34K NARC serie 8; GEO Platform GPL11051GPL) and the Affimetrix chip used by Goldberg & Harada (Ath1, GEO Platform GPL198). For the Ath1 chip we used the annotation provided by Goldberg & Harada available at For the operon chip we used the current TAIR 9.0 annotation. From these annotations all AGIs for nuclear genes were extracted and the overlap was calculated. This reference set contained 22130 genes.We used the reference set overlap of the following Goldberg/Harada datasets for comparison: GH seed  =  call all present and experiment in Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Seed; GH seed coat  =  call all present and experiment in Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Chalazal Seed Coat or Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/General Seed Coat; GH endosperm  =  call all present and experiment in Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Chalazal Endosperm or Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Micropylar Endosperm or Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Peripheral Endosperm; GH embryo  =  call all present and experiment in Arabidopsis ATH1 Array/Arabidopsis/Globular Stage/Embryo Proper.Venn diagrams were generated using the VENN diagram generator designed by Tim Hulsen at . The test for statistical significance of the overlap between two groups of genes was calculated by using software provided by Jim Lund accessible at […]

Pipeline specifications

Software tools limma, BioVenn
Databases GEO
Application Gene expression microarray analysis
Organisms Arabidopsis thaliana