GSM8901014: GFP(control), B73primaryroot, DAPseq, rep2; Zea mays; OTHER
Primary roots (~1 mm pieces) were flash-frozen in liquid nitrogen and ground into fine powder using glass beads (425–600 µm) and a Precellys 24 homogenizer (5000 rpm, 3 × 40 s). Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega). The DNA library preparation and DAP-seq experiment were performed as described by O'Malley et al. (2016).  gDNA was extracted from primary roots and 20 µg was fragmented using a sonication bath for 30 min. Sheared DNA was end-repaired using the Fast DNA End-Repair Kit (Thermo Scientific), A-tailed with Klenow Fragment (3'→5' exo-) (NEB), and Y-adapters were added with T4 DNA ligase (NEB). After final cleanup, the concentration of the DNA library was quantified using the Qubit dsDNA Assay (Invitrogen). The pENTR::NAC24 construct was LR recombined into pIX-HALO::ccdB (Bartlett et al., 2017), and the NAC24-Halo fusion protein was expressed using the TNT SP6 Wheat Germ Protein Expression System (Promega). The NAC24-HALO protein was incubated with Magne® HaloTag® Beads (Promega) for 1 h at 25°C with gentle rotation. After washing, 1 µg of gDNA library was added, and samples were incubated for 1 h at 25°C. Unbound DNA was removed with six washes, and bound DNA was eluted in Elution Buffer (QIAGEN) at 98°C for 10 min. Eluted fragments were PCR-amplified (20 cycles) to incorporate index sequences (NEBNext® Multiplex Oligos for Illumina®, NEB). PCR products were analyzed on an agarose gel, purified using the MinElute PCR Purification Kit (QIAGEN), and quantified with the Qubit dsDNA Assay (Invitrogen). Mock DAP-Seq libraries were prepared under identical conditions using GFP-Halo proteins as a negative control. Librairies were sequenced on NovaSeqX (paired-end, 150 bp reads).