2.10 ChIP assay
The ChIP assays were performed as described in our previous study (Z. Wang et al., 2020). Briefly, 15 day tobacco seedlings on MS medium were harvested, and immediately fixed in formaldehyde solution (1.0%) under vacuum infiltration for 10 min. Glycine (final concentration 0.125 M) was added to the solution under vacuum infiltration for an additional 5 minutes to stop the cross-linking reaction. The tissues were then grinded to powders in liquid nitrogen, and suspended with lysis buffer provided by the EpiQuik Chromatin Immunoprecipitation (ChIP) Kit (Epigentek). The pellet nuclei was centrifuged, and then sheared by sonication to break the DNA into 200-1000 bp fragments. The NtMYB12a-GFP fusion protein was pulled down in the strip wells with Anti-GFP antibody (Abcam, ab290). DNA fragments were released from the fusion protein, and collected for the qRT-PCR analyses. The enrichment of the genomic fragment in two tubulin beta chain genes (NtTUBB , XP_016456097.1 and NP_001312648.1) was used as a negative control. The primers used for fragment amplification are listed in Supplementary Table S3.