GSM7843113: mock,input; Triticum aestivum; OTHER
Wheat seedlings were cut into fine segment and digested. After digestion with gentle shaking, protoplasts were isolated. Protoplast transformation was carried out in PEG solution [40% (W/V) PEG 4000,2M mannitol and 0.1M CaCl2]. Transformation mixtures were agitated gently. Then, protoplasts were harvested and washed. protoplasts followed by centrifugation to remove the supernatant. Then Purified protoplasts were lysed using cold NIB for few minutes. After lysis, the cell nuclei were washed twice with 1 ml Wash Buffer. Finally, the nuclei were resuspended in 100 μL of Wash Buffer. Add 1 μL of antibody (anti-GFP antibody or IgG control antibody) to each reaction (1:100 diluted). Perform immunoprecipitation overnight at 4 °C with gentle shaking. The CUT&Tag assay was performed based on the guidance of NGS G-Type In-Situ DNA Binding profiling Library Prep Kit for Illumina. Perform the PCR reaction as follows using the NGS® Tagment Index Kit for Illumina: add 20 µL of DNA, 25 µL of 2 × Ultima Amplification buffer, 1 µL of 10-µM P5 primer X, 1 µL of 10-µM P7 primer X. Mix gently and spin briefly.