Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

RL-SAGE analysis of Zea mays leaf transcriptome

ABSTRACT: Using the RL-SAGE method (Gowda et al. 2004), a maize leaf longSAGE library (cv. inbred line B73) was constructed. Leaf tissues were harvested from 4-week old B73 plants for RNA isolation. The conditions in the growth chamber were 12 h light (500 µmol photons m-2 sec-1), 20oC at night, 26oC in the day and 85% relative humidity. A total of 44,870 unique tags (17 bases +CATG) were identified from 232,948 individual tags in the maize leaf library. This GEO Series was created by the GEO staff as part of a cleanup effort to ensure that all GEO Samples are included within a Series entry.

ORGANISM(S): Zea mays

SUBMITTER: Guo-Liang Wang

PROVIDER: E-GEOD-25994 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Robust-LongSAGE (RL-SAGE): a substantially improved LongSAGE method for gene discovery and transcriptome analysis.

Gowda Malali M Jantasuriyarat Chatchawan C Dean Ralph A RA Wang Guo-Liang GL

Plant physiology 20040301 3

Serial analysis of gene expression (SAGE) is a widely used technique for large-scale transcriptome analysis in mammalian systems. Recently, a modified version called LongSAGE (S. Saha, A.B. Sparks, C. Rago, V. Akmaev, C.J. Wang, B. Vogelstein, K.W. Kinzler [2002] Nat Biotechnol 20: 508-512) was reported by increasing tag length up to 21 bp. Although the procedures for these two methods are similar, a detailed protocol for LongSAGE library construction has not been reported yet, and several techn ...[more]

PMID: 15020752

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Project description:Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression (DGE) profiles using Illumina’s high-throughput sequencing technology and the newly-assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in determinacy of axillary meristems. The RA3 gene encodes a trehalose-6-phosphate-phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of DGE libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20-21nt reads with frequencies spanning four orders of magnitude. Unique sequence tags were anchored to 3´-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of non-redundant signature tags to the maize genome, which associated with 37,117 working gene models and un-annotated regions of expression. 66% of maize genes were detected at ? nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis and rice in functional analyses of differentially expressed maize genes. Results from this study provide a basis for analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene. 3' tag-based DGE libraries were generated using Illumina 1G technology. Libraries were prepared from 2mm ear samples and represent: 3 biological replicates of wild-type B73, 3 biological replicates of ramosa3 (ra3) mutants, and 1 technical replicate of a ra3 sample. Each sample was sequenced in a single lane of a flow cell. Each lane includes library preparations from both DpnII and NlaIII digests for that sample. Sequences from DpnII digests start with GATC and those from NlaIII digests start with ATG.

Project description:To gain novel molecular insights into quantitative late blight resistance, we performed a high-resolution quantitative analysis of gene expression using potato cultivars with contrasting SNP alleles at the StAOS2 locus associated with maturity corrected resistance (MCR). SuperSAGE samples were generated from uninfected and infected plants of the selected genotypes under controlled environmental conditions. Genotypes were pooled to reduce the influence of the genetic background on the transcriptome. Nine SuperSAGE samples were prepared from artificially inoculated plants in a growth chamber using total RNA of the pooled 14, 6 and 9 genotypes in groups A1, A2 and B2, respectively, at three infection time points T0, T1 and T2. Combining the tag counts of both NlaIII and DpnII libraries resulted in 1.1 to 6.2 million tags per sample. Of total 266361 unique tags (unitags), 52.6% matched to the potato genome sequence when up to three mismatches per 26 base pairs were allowed, and 23.3% matched without mismatch. Fifteen pair wise comparisons were performed between the nine SuperSAGE samples to identify transcripts that were differentially expressed in response to infection (six comparisons) or between three genotype pools at the infection time points T0, T1 and T2 (nine comparisons). The number of unitags per comparison ranged from 127 000 to 182 000 (average 158 000). Between 2100 and 11800 tags were differentially expressed in pair wise comparisons, depending on arbitrary cut-off p-values for a significant difference. The highest number of differences was observed for the comparison between genotype pools A1 and A2 one day after infection (A1-T1 vs A2-T1), and the lowest between genotype pools A2 and B2 two days after infection (B2-T2 vs A2-T2). The number of differences in response to infection and between genotype pools even before infection (T0) was in the same order of magnitude. Based on the annotations in the DFCI potato gene index, in the potato genome and in few cases by BLASTX searches against the protein database at NCBI, transcripts that showed reproducible differential expression over the infection time course or between genotype pools A1, A2 and B2 were grouped in 16 functional categories, with overlaps between categories. Genes with genotype dependent, constitutive differential expression provide excellent targets for developing novel diagnostic markers for breeding cultivars with improved quantitative resistance to late blight and possibly other biotic and abiotic stresses. Relevant in this respect appear, besides numerous genes of unknown or ill-defined function, genes with known function involved in stress responses, photosynthesis, protein biosynthesis, protein degradation via the 26S proteasome, transport of proteins, lipids, ions and other small molecules, cell wall structure and many others. Eighteen SuperSAGE libraries were constructed based on nine leaf samples from one infection experiment. For each time point (T0, T1 and T2) one leaflet each of 14, 6 and 9 SL genotypes in genotypic groups A1, A2 and B2, respectively, were pooled. Frozen pooled leaf tissue was powdered in a CryoMill. SuperSAGE libraries were generated at GenXPro GmbH (Frankfurt, Germany) essentially as described (Matsumura et al. 2010). To prevent amplification biases the TrueQuant technology was applied as described by B. Rotter (Patent application Nr. WO2009152928). Besides NlaIII (recognition site: 5M-bM-^@M-^Y-CATG-3M-bM-^@M-^Y), DpnII (recognition site: 5M-bM-^@M-^Y-GATC-3M-bM-^@M-^Y) was used as second anchoring enzyme, to capture transcripts without a NlaIII site. Therefore, the 26 bp tags carry either CATG or GATC at their 5M-bM-^@M-^Y end. The libraries were pooled and sequenced by Solexa/Illumina technology (Illumina, Inc., USA).

Dataset Information

RL-SAGE analysis of Zea mays leaf transcriptome

Publications

Robust-LongSAGE (RL-SAGE): a substantially improved LongSAGE method for gene discovery and transcriptome analysis.

Similar Datasets

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