GSM8291347: ZmWUS1, DAP-seq; Zea mays; OTHER
genomic DNA was extracted from the leaves of 14 day-old B73 seedlings using a phenol:cholorform:Iso amyl alcohol procedure. 5ug of extracted DNA was sheared in a Covaris S2 sonicator to 200bp fragments. The fragmented DNA was then end repaired using the END-IT DNA repair kit (Lucigen) per the manufacturer's recommendations and cleaned using a QiaQuick PCR clean-up method (Qiagen). Eluted DNA was then A-tailed using Klenow exo- (NEB) for 30min at RT. Samples were cleaned using a QiaQuick PCR-clean-up method. Eluted DNA was ligated to truncated Illumina Y-adapters overnight at 16 degrees C using T4 DNA ligase (NEB). Samples were heat inactivated for 10 min at 70 degrees C followed by a 1:1 bead clean-up using AmpureXP beads (Beckman-Coulter). Libraries were quantified using the Qubit HS kit. 1ug of this library was then incubated with in vitro expressed HALO-WUS1 protein bound to 10ul of MagneHALO beads (TNT rabbit reticulocyte expression system and MagneHALO beads, Promega) generated from 1ug of pIX-HALO-WUS1 plasmid. Unbound DNA was washed away using six washes of phosphate buffered saline (PBS), and bound DNA was eluted from the beads using 30ul of 10mM Tris (EB) and heating to 98 degrees C for 10 min. DNA was barcoded and enriched with 19 cycles and Illumina primers prior to sequencing on a NextSeq500 with 75bp Single End reads.