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SRX4012511: GSM3121162: WT 2; Arabidopsis thaliana; RNA-Seq
1 ILLUMINA (Illumina HiSeq 4000) run: 26.1M spots, 7.7G bases, 3.6Gb downloads

Submitted by: NCBI (GEO)
Study: EML histone readers prevent seed development without fertilization
show Abstracthide Abstract
EML1 and EML3 were previously shown to be histone readers involved in plant-pathogen interactions. To learn more about the developmental function of EML1 and EML3, we generated eml1 eml3 double mutant and showed that it had specific seed developmental phenotypes, including a capability to develop seed without fertilization. Next, we analyzed the mRNA expression of genes in the eml1 eml3 double mutant and compared it to its wild type. Differentially expressed (DE) genes in the mutant were identified and compared with DE of the mutants known to be involved in regulating seed development and in fertilization-independent endosperm development. Our results suggest that some targets are shared between EML histone readers and known regulators of seed development, such as MEA. Auxin response seems to be affected in both types of mutants. However, unlike MEA, EML proteins regulate auxin responsive genes not only in the endosperm, but also in the embryo. This capability makes EML proteins very good candidates for engineering apomictic seeds. Overall design: 3 eml1,eml3 double mutant samples and 3 WT samples
Sample: WT 2
SAMN09001004 • SRS3234070 • All experiments • All runs
Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA extracted from WT and eml1-2 eml3-4 siliques (1-5 DAP) were treated with DNase I (Life Tecnologies) twice to ensure no genomic DNA contamination. Two micrograms of total RNA was subjected to two rounds of hybridization to oligo(dT) beads (Illumina). The poly(A)-enriched mRNA was fragmented by heating at 94°C for 5 min in fragmentation buffer supplied by Illumina, followed by ethanol precipitation. The fragmented mRNA was added to 1× first-strand buffer (2.5 mM deoxynucleotide triphosphate, 10 mM DTT, RNaseOUT, and random primers mix). After adding Superscript II, first-strand cDNA synthesis was performed at 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. The second-strand cDNA synthesis was performed in GEX Second Strand Buffer (25 mM deoxynucleotide triphosphate, 1 μL RNase H, and 5 μL DNA Pol I for 2.5 h at 16°C) and purified by a PCR purification kit (Qiagen). After end repair and A-base addition, adapter ligation was performed using NEBNext system according to the manufacturer's instructions (NEB). We used Index 25, 27 and 4 or Index 6, 7 and 12 for WT or eml1-2 eml3-4, respectively. Ligated fragments were subjected for selection of fragments of ∼300 bp in length, followed by PCR amplification (10 cycles). PCR products were purified with Agencourt AMPure (Beckman Coulter). Library DNA was checked for concentration and size distribution in an Agilent Bioanalyzer before being subjected to Illumina sequencing using the OSUCCC Nucleic Acid Shared Resource (The Ohio State University).
Experiment attributes:
GEO Accession: GSM3121162
Runs: 1 run, 26.1M spots, 7.7G bases, 3.6Gb
Run# of Spots# of BasesSizePublished


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