GSM8032893: ZmICE1a-mutant-biorep2; Zea mays; OTHER
About 100 mg of endosperm powder was resuspended in pre-cold nuclear isolation buffer A (10 mM Tris-HCl (pH 8.0), 10 mM KCl, and 0.5 mM spermidine) and filtered through Miracloth (Millipore, Burlington, MA, USA; cat. no. 475855-1R). Cell nuclei were extracted using pre-cold nuclear isolation buffer B (10 mM Tris-HCl (pH 8.0), 10 mM KCl, 0.5 mM spermidine, 0.5 % Triton X-100, and 1X cocktail) and then washed three times. Cell nuclei were incubated with ZmICE1a antibody (final concentration, 20 μg/mL) for 2 h at room temperature. Goat anti-rabbit IgG (secondary antibody) was added to each sample (final concentration, 10 μg/mL). After washing the samples three times, they were incubated with pA-Tn5 transposome at room temperature for 1 h. The samples were washed three times to remove unbound pA-Tn5 transposome and then incubated in tagmentation buffer (10 mM TAPS-NaOH (pH 8.5), 10 mM MgCl2, 10% (v/v) DMF, 0.5 mM spermidine, 0.05% (v/v) digitonin, and 1% (v/v) plant cocktail) at 37°C for 1 h. The reactions were stopped, and then the DNA was extracted from the samples. Finally, CUT&Tag-DNA libraries were constructed and sequenced using an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) by BerryGenomics (China).