GSM9736686: WRKY2-HA, young leaves, HA (ChIP); Solanum lycopersicum; ChIP-Seq
1.00g young leaves tissues from SlWRKY2-HA and SlPIF4-HA tomato seedlings at the five-leaf stage under normal conditions are harvest and fixed with formaldehyde to covalently crosslink proteins to DNA, and the crosslinking reaction is subsequently quenched with glycine. The frozen tissues were then pulverized to a fine powder within a liquid nitrogen-prechilled mortar. Following cell lysis in a buffer consisting of 250 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% (v/v) Triton X-100, 0.10% Na-Deoxycholate (30990-25G; Sigmal-Aldrich, USA), 10mM DTT, chromatin is sheared by sonication into fragments of 200-500 bp, and the fragment size distribution is verified via 2% agarose gel electrophoresis. The solubilized chromatin is pre-cleared and then incubated with anti-HA antibodies (ab9110; abcam, USA) directed against the target protein, followed by immunoprecipitation using protein A/G magnetic beads (26162; Fisher Scientific, USA). An input sample is reserved as a control. The beads are subjected to stringent washes in order to eliminate non-specific binding using low salt wash buffer (20 mM Tris-HCl, pH 8.0; 150mM NaCl, 2mM EDTA, pH 8.0, 0.1% SDS, 1% (v/v) Triton X-100), high salt wash buffer (20 mM Tris-HCl, pH 8.0; 500mM NaCl, 2mM EDTA, pH 8.0; 0.1% SDS, 1% (v/v) Triton X-100) and LiCl wash buffer (250mM LiCl, 1mM EDTA, pH 8.0; 1% NP-40, 1% Na-Deoxycholate), after which crosslinks are reversed by incubation at 65℃ in a elution buffer (10mM Tris-HCl, pH 8.0; 1mM EDTA, pH 8.0; 1% SDS). The released DNA is purified using phenol-chloroform extraction. Enrichment at known binding sites is assessed by quantitative PCR (qPCR) with specific primers. The immunoprecipitated DNA is subsequently used for library construction, which involves end repair, adapter ligation, and PCR amplification. Library quality and quantity are evaluated using Qubit (Q33238; Thermo Fisher Scientific, USA), Bioanalyzer, and qPCR.