GSM8143916: MYB67 replicate 2; Arabidopsis thaliana; OTHER
Genomic DNA(gDNA)libraries for Arabidopsis were created following protocols in Bartlett et al. andO'Malleyet al. with minor modifications. 10µg of Arabidopsis gDNA extracted from leaf tissue was fragmented to∼200bp ina CovarisS2sonicator. The fragmented gDNA was end-repaired using the End-It DNA End-Repair Kit (Biosearch Technologies, ER81050), A-tailed usingKlenow Fragment (3–5′exo) (NEB, M0212L), and the adaptor was ligatedusingthe truncatedIllumina TruSeq Y-adaptor.The adapter-ligatedlibrary was confirmed withRTqPCRas described in Bartlett et al. The MYB67 cDNA was TOPO cloned into a pIX-Halo plasmid (ABRC, stock # CD3-1742) per manufacturer's instructions (ThermoFisher, 450218). This construct, along with an empty plasmid containing only the Halo tag, were incubated with the TNT SP6 Coupled Wheat Germ Extract System to produce Halo-MYB67 or Halo only proteins, respectively (Promega, PRL4130). 40µl of the resulting protein extracts were mixed with 10µl Magne HaloTag Beads (Promega, G7281) and 30ul of wash buffer (WB1; PBS + 0.005% NP40) on a rotator for 1h at room temperature. The beads with protein were then washed with WB1 as described in Bartlett et al. and then incubated with 100ng of the adapter-ligated gDNA library and also washed with WB1 for 1hr. The beads were then washed with WB1 as described in Bartlett et al. to remove unbound DNA fragments and then incubated at 98°C for 10min to denature the protein and release the bound DNA fragments. To amplify the released DNA, PCR reactions were prepared using Phusion DNA Polymerase (NEB, M0530S), a universal primer, and a unique Illumina index primer, to allow multiple samples to be pooled in one sequencing run. PCR products were then size-selected using Sera-Mag beads (Sigma-Aldrich, GE65152105050250) at a 1.0x ratio. The purified DNA libraries were measured by the Qubit HS ds DNA Assay Kit (ThermoFisher, Q32854) and sequenced on the Nextseq2000 with 100 cycles and 50bp sequence length.