title: GSM9444363: GRF5 DAP-seq, Input control; Arabidopsis thaliana; OTHER
library_construction_protocol: Root tissues from low nitrate-treated Arabidopsis seedlings were harvested, ground into powder in liquid nitrogen, and genomic DNA was isolated using the Qiagen DNeasy Plant Mini Kit according to the manufacturer's instructions; 2. Recombinant GRF5 protein was expressed and purified from Escherichia coli using a His-tag affinity purification system, followed by verification of protein purity via SDS-PAGE. DAP-seq libraries were prepared as follows: 1. Purified GRF5 protein was incubated with Arabidopsis root genomic DNA fragments (200-500 bp) to form protein-DNA complexes; 2. Protein-bound DNA was enriched via immunoprecipitation with anti-GRF5 antibody; 3. Enriched DNA fragments were used as input for library construction with the KAPA HyperPlus Library Preparation Kit (Roche); 4. Libraries were amplified with 12 PCR cycles, size-selected (250-450 bp) using Agencourt AMPure XP beads, and validated by Agilent High Sensitivity DNA Analysis.