GSM5765982: bHLH-6A-1; Triticum aestivum; OTHER
Genomic DNA was extracted from leaves using Plant DNAzol Reagent(invitrogen). RNA was extracted using TRIzol. Genomic DNA was fragmented. And then end repaired using the End-It kit(Lucigen) and A-tailed using Klenow(3'-5' exo-; NEB). Truncated Illumina Y-adapter was ligated to DNA using T4 DNA Ligase(Promega). Full length TF was cloned into pIX-Halo vector. Halo-tagged TF was expressed in vitro using TNT SP6 Coupled Wheat Germ Extract System(Promega). Halo-TF was immobilized by Magne HaloTag Beads(Promega) and then incubated with the DNA library. TF specific binding DNA was eluted for 10 min at 98°C and amplified with indexed Illumina primer using Phanta Max Super-Fidelity DNA Polymerase(Vazyme). Meanwhile, to capture background DNA which captured by Halo, pIX-Halo vector without TF cloned was expressed and incubated with the DNA library as well. The PCR product was purified using VAHTS DNA Clean Beads(Vazyme).