An optimised chromatin immunoprecipitation (ChIP) method for mature leaves of Nicotiana benthamiana to study epigenetic landscape of an allotetraploid plant
- Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Australia.
- School of Biological Sciences, The University of Queensland, Brisbane, Australia.
Histone modifications are involved with mitotic and meiotic inheritance of active and silent gene variants in plants. These epigenetic modifications play an important role in physiological and phenotypic features of a plant. Nicotiana benthamiana is an important biotech tool and a model plant for economically important crop family Solanaceae. To study the epigenetic landscape of this allotetraploid plant, we optimised a ChIP protocol using mature leaves of N. benthamiana. We first tested a number of published ChIP protocols, however high concentration of starch was unfavourable in obtaining good quality of nuclei. In this study, we optimised nuclei isolation, storage and chromatin shearing steps to develop a consistent ChIP method for N. benthamiana. We found that Covaris M220 was more efficient compared to conventional DNA shearing using BioRuptor. ChIP was performed using antibodies for H3K4me2/3 and H3K9me2 histone modifications and success of protocol was determined by PCR and next generation sequencing. This optimised method is applicable to ChIP of starchy tissues followed by high-throughput DNA sequencing to map epigenetic landscapes.