GSM8808040: HSFA1_DAP, rep3; Marchantia polymorpha; OTHER
For protein expression and purification, the coding sequences (CDS) for Marchantia HSFA1 and the N-terminal of HaloTag, along with the sequences for the SP6/T7 promoter and T7 terminator, were synthesized by Gene Universal, USA. These sequences were sub-cloned using BbsI into pAGM9121 as level 0 modules and further assembled via Golden Gate cloning into the level 1 expression vector pICH47732 to generate an SP6/T7 promoter-driven CDS with a N-terminal HaloTag and a T7 terminator. Recombinant proteins were expressed using the TNT SP6/T7 High-Yield Wheat Germ Protein Expression System (Promega) following the manufacturer's protocol. 2.5 μg of plasmid was used to translate protein in vitro at 30°C for 2 hours using a reagent mixture. Protein purity and integrity were confirmed by analyzing 10% of the final mixture via SDS-PAGE confirmed protein purity and integrity. For genomic DNA, approximately 1 g of 14-day-old Marchantia gemmalings were collected and homogenized under liquid nitrogen. Genomic DNA was extracted using the phenol/chloroform method and sheared mechanically to an average size of 100-500 bp. The DNA fragments were end-polished, A-tailed, and ligated with a truncated Y-shaped adapter to prepare them for the protein-DNA binding assay. In vitro translated proteins were incubated with HaloTag beads (Promega) for 1 hour at room temperature and purified using a magnetic separator with three washes in binding buffer (PBS with 0.1% NP-40). The purified beads-TF complexes were then incubated with the sheared, adapter-ligated DNA in the binding buffer to facilitate protein-DNA interactions. The mixture was incubated at room temperature for one hour to form TF-DNA complexes. Bead-TF-DNA complexes were captured using a magnetic base and washed three times to remove non-specifically bound DNA. Bound DNA was eluted from the beads by heating the mixtures to 98°C for 10 minutes. PCR was used to enrich the protein-bound DNA, incorporating a unique index for each sample to prepare them for multiplex sequencing.