Fig. 3: Analysis of the effects of different hybridization buffers and cross-linking methods on decross-linking of chromatin.

a Analysis of the effects of SDS concentration and different buffers on decross-linking of chromatin. MS and LS hybridization buffer [containing 10% (v/v) formamide supplied with 0, 0.5%, and 1% (w/v) SDS] were used in hybridization at 37 °C for 4 h. To analyze decross-linking, the chromatin was cross-linked with 3% (w/v) formaldehyde, and treated at 90 °C for 2 min for denaturation before hybridization. b Analysis of the effects of different crosslinking methods on preventing decross-linking of chromatin during heat and hybridization. The chromatin was cross-linked with 1%, 3%, or 3% (w/v) formaldehyde supplement with 20 mg L⁻¹ NiSO₄, and incubated in LS buffer [with 10% (v/v) formamide, 0.5% (w/v) SDS] at 90 °C for 2 min to denature the chromatin, and then hybridized for 4 h. After heating and hybridization, the decross-linked DNA was quantified using qPCR. The y-axis indicates the CT value difference (i.e., ΔCt), ΔCt = Ct (decross-linked DNA before heat) −Ct (decross-linked DNA after hybridization). Increased ΔCt value indicates increased decross-linking of DNA and protein. Three replicates (sample size of 50 seedlings) were performed (n = 3 independent experiments). Error bar indicates standard deviations of the mean measurements. Multiple comparisons of means were performed using Tukey’s test, and different letters represent significant differences among treatments (P < 0.05). The promoter region of AtCAT3 used for the qPCR evaluation was site 1 and is shown in Fig. 7l and Supplementary Data 4.