4.7. DNA Affinity Purification Sequencing (DAP-Seq) Sampling

DAP-seq was performed according to Bartlett et al. (2017) [76]. First, genomic DNA (gDNA) was extracted from mature seeds of rice. Then a gDNA DAP-seq library was prepared by attaching a short DNA sequencing adaptor to the purified and fragmented gDNA. The adapter sequences were truncated Illumina TruSeq adapters; the TruSeq Universal and Index adapters corresponded to the DAP-seq Adapter A and Adapter B. The DAP gDNA library was prepared using a kit from NEBNext^® DNA Library Prep Master Mix Set for Illumina^® (NEB, #E6040S/L, Ipswich, MA, USA). OsbZIP09 was fused to the HaloTag using a kit from pFN19K HaloTag T7 SP6 Flexi Vecto (Promega #G184A). OsbZIP09 fused to HaloTag was expressed using a TnT SP6 High-Yield Wheat Germ Protein Expression System (L3260, Promega, Madison, WI, USA), and was purified using Magne HaloTag Beads (G7281, Promega). The Magne HaloTag Beads and OsbZIP09-HaloTag mixture were incubated with 500 ng DNA library in 40 μL PBS (Phosphate Buffered Saline) buffer with slow rotation in a cold room for 1.5 h. The beads were washed five times with 200 μL PBS + NP40 (0.005%), resuspended in PBS buffer, the supernatant was removed, and 25 μL EB buffer was added and samples were incubated for 10 min at 98 °C to elute the bound DNA from the beads. The correct DAP-seq library concentration to achieve a specific read count was calculated on the basis of on library fragment size. Negative-control mock DAP-seq libraries were prepared as described above, without the addition of protein to the beads.