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SRX864544: GSM1600238: Control line, H3K4me3; Brassica napus; ChIP-Seq
1 ILLUMINA (Illumina HiSeq 2500) run: 13.2M spots, 2.6G bases, 1.6Gb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: Selection for improved energy efficiency and drought tolerance in canola results in distinct transcriptome and epigenome changes [ChIP-Seq]
show Abstracthide Abstract
Integrated breeding strategies are used to increase both the yield potential and stability of crops. Most of these approaches have a direct genetic basis. The utility of epigenetics in breeding to improve complex traits such as yield and stress tolerance is not clear. A better understanding of the status of the epigenome and its contribution to the agronomic performance would help in developing strategies to incorporate the epigenetic component of complex traits in breeding,Starting from isogenic canola lines, epilines were generated by selecting recursively during three generations for lines with a higher energy use efficiency and drought tolerance. These epilines were more energy use efficient, drought tolerant, high nitrogen use efficient, and higher yielding under suboptimal conditions. Moreover, these characteristics were transgenerational inheritable. Transcriptome comparison with a line selected for energy use efficiency only revealed common differentially expressed genes related to the onset of signaling events regulating stress tolerance. Genes related to salt, osmotic, abscisic acid and drought were specifically differentially expressed in the drought tolerant epilines. The status of the epigenome, scored as differential trimethylation of lysine 4 of histone 3, supports the energy use efficient and drought tolerant phenotype by facilitating transcription of the genes that are found to be differentially expressed.From these results it can be concluded that the epigenome can be shaped by selection to increase yield and stress tolerance. This acquired knowledge will support further development of strategies to incorporate epigenetics in breeding. Overall design: To investigate the epigenetic effect on histone mark distribution of the EUE/PEG selection we performed ChIP-seq analyses. Native ChIP using an anti-H3K4me3 and no antibody (background control) was done on PEG1 and control plants.
Sample: Control line, H3K4me3
SAMN03327471 • SRS835398 • All experiments • All runs
Organism: Brassica napus
Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Selection: ChIP
Layout: PAIRED
Construction protocol: The protocol for native chromatin immunoprecipitation was adapted from Bernatavichute et al., 2008. Leaf 4 material of 26 day-old wild-type and PEG1 plants was harvested, frozen in liquid nitrogen and ground to powder (1.5 g). Plant tissue was resuspended in 15 ml of HBM buffer (25 mM Tris-Cl pH 7.5, 440 mM sucrose, 10 mM MgCl2, 0.1% Triton-X, 10 mM beta-mercaptoethanol, 2 mM spermine, 1 mM PMSF, 1 ug/ml pepstatin, 1 ug/ml aprotinin and 1 ug/ml leupeptin), homogenized and filtered twice through Miracloth (Calbiochem). After spinning at 3000 rpm for 5 min (4°C, SS-34, Sorvall), the pellet was resuspended in 5 ml of NIB buffer (20 mM Tris-Cl pH 7.5, 250 mM sucrose, 5 mM MgCl2, 5 mM KCl, 0.1% Triton-X, 10 mM beta-mercaptoethanol, 1 mM PMSF, 1 ug/ml pepstatin, 1 ug/ml aprotinin and 1 ug/ml leupeptin), applied to a 15/50% Percoll (GE Healthcare) gradient in NIB and spun 2000 rpm for 20 min (4 C, SS-34, Sorvall). Isolated nuclei were washed three times in NIB buffer and flash frozen in liquid nitrogen in HBC buffer (25 mM Tris-Cl pH 7.5, 440 mM sucrose, 10 mM MgCl2, 0.1% Triton-X, 10 mM beta-mercaptoethanol, 20% glycerol). Nuclei from each preparation were diluted 2-fold in digestion buffer (20 mM Tris-Cl pH 7.5, 0.22 M sucrose, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 5 mM Na butyrate), spun 600 g for 5 min (4°C, Eppendorf, 5415R) and resuspended in 0.25 ml digestion buffer for Micrococcal Nuclease (30 U, Affymetrix) digestion for 10 minutes at 37 °C stopped with 10 mM EDTA. Mononucleosomes were released by treating nuclei with 0.1% Triton-X for 2 hours in the cold, and then pelleting the debris by centrifuging at 10000 rpm for 5 min (4°C, Eppendorf, 5415R). The supernatants were diluted to 0.5ml in incubation buffer (20 mM Tris-Cl pH 7.5, 50 mM NaCl, 5 mM EDTA, 0.1% Triton, 20 mM Na butyrate, 0.1 mM PMSF, 1 ug/ml pepstatin, 1 ug/ml aprotinin and 1 ug/ml leupeptin) and overnight incubated in the cold with and without 2.5 ug of the anti-H3K4m3 antibody (#07-473, Millipore). 50 ul of Dynabeads Protein G (Invitrogen) were applied, incubated for 3 hours in the cold and washed three times with 500 ul wash buffer (50 mM Tris-Cl pH 7.5, 10 mM EDTA, 5mM Na butyrate, 0.1 mM PMSF, 1 ug/ml pepstatin, 1 ug/ml aprotinin and 1 ug/ml leupeptin) with increasing concentrations of NaCl (50 mM, 100 mM and 150 mM), subsequently. Final wash was done in TE buffer (10 mM TrisCl pH8, 500 mM NaCl, 1 mM EDTA) and immunocomplexes were eluted and reverse crosslinked by incubation overnight at 65°C in 0.25 ml 10 mM TrisCl pH8, 1 mM EDTA, 0.5 M NaCl, 1% SDS + 1 µl RNAse A (100 mg/ml). Eluates were incubated with 100 µg Proteinase K for 2 h at 42°C and the DNA extracted by phenol/chloroform/IAA followed by purification using Qiaquick PCR purification kit and DNA quantification by the Quant-iT dsDNA High Sensitivity kit (Invitrogen). Standard Illumina Protocol
Experiment attributes:
GEO Accession: GSM1600238
External link:
Runs: 1 run, 13.2M spots, 2.6G bases, 1.6Gb
Run# of Spots# of BasesSizePublished


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