GSM5746466: A2-A3; Arabidopsis thaliana; OTHER
Genomic DNA was isolated from young seedlings of Col-0 using CTAB method, and digested with RNase A and cleaned using phenol: chloroform: isoamylalcohol. Isolated genomic DNA was sonicated to an average length of 200 bp in Covaris sonicator and quantified. After sonication, fragmented genomic DNA was end prepared and adaptor ligated according to the protocol of  NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645), and purified using NaOAc and ethanol. The DNA was diluted in Elution buffer (10 mM Tris-HCl, pH 8.5) and measured using Qubit dsDNA HS Assay Kit. 200-300 ng prepared DNA was used for each binding reaction. cDNA sequences of HSFA1b, HSFA2 and HSFA3 were subcloned into pIX-HALO. HALO-tagged HSFs were expressed in the TNT wheat germ expression system (Promega). Different combinations of HSFs or HALO (negative control) were incubated with prepared DNA overnight at room temperature, respectively. For 37°C treatment, the samples were treated at 37°C for 45 min before overnight incubation at room temperature. Washing and DNA recovery steps were as described (Bartlett et al., 2017). The recovered DNA was amplified using Q5 Master Mix and different index primers in NEBNext® Ultra™ II DNA Library Prep Kit, then pooled and size selected (200-700 bp) by gel fractionation. 10 nM DNA library was sequenced using NextSeq 500/550 Mid output Kit (300 cycles), 150 bp SE reads.