GSM8978186: morc-A1-2; Oryza sativa Japonica Group; OTHER
Seedling were cut into small pieces and the nuclei were separated. Nuclei were obtained per sample for subsequent experiments, washed with wash buffer, and immobilized to concanavalin A-coated beads (Vazyme, TD903) with incubation at room temperature for 10 min. The bead-bound nuclei were incubated in 200 μL of primary antibody buffer with 1:50 anti-EMF2b or anti-ALKBH1 antibody dilution at 4 °C by rotating overnight. The next day, the primary antibody buffer was removed and cells were washed with 800 μL of dig-wash buffer. After washing, antibody-incubated nuclei were resuspended in 200 µL of dig-wash buffer with a 1:100 dilution of secondary antibody (Vazyme, TD903) and incubated at room temperature with gentle rotation for 1 h. To eliminate binding antibodies, nuclei were washed three times with 800 µL dig-wash buffer. After a brief wash with dig-wash buffer, nuclei were resuspended in 200 µL of dig-300 buffer and incubated at room temperature for 1 h with gentle rotation. pA-Tn5-bound nuclei were washed with 800 µL of dig-300 buffer three times, then tagmentated at 37 °C for 1 h. 15 mM EDTA, 500 µg/ml proteinase K, and 0.1% SDS were added after tagmentation to terminate tagmentation and degrade protein. Genomic DNA was extracted and purified with VAHTS DNA Clean Beads (Vazyme, N411-01). Purified genomic DNA was amplified using the universal i5 primer and barcoded i7 primer (Vazyme, TD202) using Vazyme Master Mix (Vazyme, TD903). The library PCR products were cleaned with Agencourt AMPure XP beads (Beckman Coulter, A63881).