E-MTAB-1663:10dCRH3K27me3
Salt priming A. thaliana ChIP-Seq
Salt priming A. thaliana ChIP-Seq
10dCRH3K27me3_extract
Arabidopsis thaliana (Col-0) seeds  were germinated on vertical agar plates containing 70 ml of nutrient solution containing 0.5 mM CaCl2, 0.25 mM MgSO4, 2 mM KNO3, 1 mM NaCl, 0.5 mM NaH2PO4, 42.5 uM Fe(III)Na-EDTA, 1.8 uM MnSO4, 45 uM H3BO3, 0.38 uM ZnSO4, 0.015 uM (NH4)6Mo7O24, 0.16 uM CuSO4, and 0.01 uM CoCl2, supplemented with 3% sucrose and 1% agar. Plants were grown at 22  degreesC  with a 10/14 h light/dark period and a light intensity of 120 uMol m-2s-1. When seedlings had reached the 4-leaf stage the priming treatment was applied directly to the roots. In a first experiment (24h short memory) roots and shoots were harvested immediately after the priming treatment . The tissue was fixed and stored at -80  degreesC. In a second experiment (10d long memory), primed and non-primed plants were transferred to hydroponic culture, and allowed to grow in (salt-free) nutrient solution  for another 10 days before harvesting root and shoot material of the mature plants The material was again fixed and stored at -80  degreesC ('10 d' samples). The priming treatment consisted in application of 5 ml of growth medium supplemented with 50 mM NaCl directly to the roots of the plate-grown seedlings. Control plants were treated in the same way using nutrient medium without NaCl added. Plants were incubated with the priming (or control) solution for 24 hours. Chromatin extraction and immunoprecipitation (ChIP) were carried out following a published protocol (Saleh A. et al. 2008,  Plant Cell, 20:568-579). In brief, tissue samples were incubated in 1% (w/v) formaldehyde for 15 min under vacuum. Cross-linking was stopped by adding 125 mM glycine, and tissues were rinsed, blotted dry and frozen. Chromatin extracts were incubated with antibodies against the following four histone modifications: H3K4me2 (Diagenode pAb-035-050), H3K4me3 (Diagenode pAb-003-050), H3K9me2 (17-681, Millipore) or H3K27me3 (Diagenode, pAb-069-050). Immunoprecipitated chromatin-DNA (IP-DNA) or input chromatin-DNA was reverse cross-linked and residual protein was removed by proteinase K treatment. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. ChIP DNA from three independently grown and treated plant batches were pooled for sequencing. A DNA library was prepared using the ChIP-SEQ Sample Prep Kit (Illumina), according to the manufacturer's protocols. Briefly, the overhangs were converted into phoshorylated blunt ends and 3'-ends adenylated, followed by adapter ligation. The library was size selected on an agarose gel and amplified by PCR.
University of Glasgow
E-MTAB-1663:10dCRH3K27me3
Experimental Factor: growth condition
no salt treatment and subsequent growth without salt
Experimental Factor: immunoprecipitate
anti-H3K27me3
Experimental Factor: sampling time
240 hour