Anti h3k9ac

Two grams of rice seedling leaves was cross-linked by 1% formaldehyde and used for chromatin extraction. After sonication, chromatin fragments were incubated with antibody (anti-H3K9ac, anti-Kbu, and anti-Kcr)-coated beads (Invitrogen/Life Technologies; 10001D) overnight. Specificity of anti-Kbu and anti-Kcr was tested by dot blots (Additional file 1: Figure S8). After extensive washing, immunoprecipitated chromatin was de-cross-linked and retrieved for qPCR or sequencing. anti-H3K9ace (07-352; Millipore), anti-butyryllysine (PTM-301; PTM Biolabs), and anti-crotonyllysine (PTM-501; PTM Biolabs) antibodies were used.

Approximately 1.5 g of 10-day-old seedlings were collected without cross-linking and stored at −80 °C until use. ChIP was executed according to the previous study (Qiu et al, 2019 (link)), and the anti-HDA9 antibody was produced by ABclonal Biotech. The sonicated chromatin extractions were immunoprecipitated with antibodies anti-H3K9ac (Abcam), anti-H3K27ac (Abcam), and anti-HDA9 for Col-0, dana1-2 and dip1-1 plants at 4 °C for 4 h on a rotation mixer. For DIP1 OE (dana1-2) plants, antibodies mouse IgG (Cell Signaling Technology) and anti-GFP (Roche) were applied. Dynabeads Protein A (Invitrogen) was applied to capture the immunocomplexes with antibodies anti-H3K9ac and anti-H3K27ac, and Dynabeads Protein G (Invitrogen) was applied to capture the immunocomplexes with antibodies anti-HDA9, mouse IgG and anti-GFP. After reverse cross-linking and proteinase K (Merck Millipore) digestion, DNAs were extracted with phenol-chloroform and the precipitated with ethanol. Primers used for ChIP-qPCR are listed in Appendix Table S1.