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.