GSM5410366: TPR1-GFPeds1_aGFP_r3 (ChIP-Seq); Arabidopsis thaliana; ChIP-Seq
ChIP was performed on 2 g leaf tissues of 6-week-old plants according to Gendrel et al. (2005) with modifications (Birkenbihl et al., 2012). Leaf samples were cross-linked twice by infiltration with 1% formaldehyde under a vacuum for 2x 7.5 minutes. Nuclei were disrupted by sonication 10 times for 30 s each and 30 s of cooling in a Bioruptor (Diagenode). IPs were performed using α-GFP (Ab6556, Abcam) antibodies and Protein A agarose beads (Sigma). After elution of immune complexes, cross-linking was reversed with an overnight incubation at 65°C in 200 mM NaCl followed by proteinase K digestion. DNA was extracted with phenol-chloroform and precipitated with ethanol in the presence of 0.3 M sodium acetate (pH=5.2) and 10 μg/mL glycogen (overnight at -20°C). After centrifugation, DNA pellets were washed with 70% ethanol, air-dried and resuspended in water before PCR. MiniElute PCR Purification Kit (Qiagen, Germany) was used for cleaning ChIP DNA and two rounds of in vitro transcription by T7 RNA polymerase followed according to a linear DNA amplification (LinDA) protocol (Shankaranarayanan et al., 2011). Sequencing libraries were barcoded and generated using Ovation® Ultralow DR Multiplex system (NuGEN Technologies Inc.) including a 16-cycles PCR amplification. Libraries were size-selected (200-350 bp) with an agarose gel extraction using the MinElute Gel Extraction Kit (Qiagen, Germany).