DAP-seq of CBF12C: stem base 6h NAA
Barley stem base samples for DAP-seq were collected before treatment (0h) and 3h after 1-NAA treatment for HvNAC013 and 6h after 1-NAA treatment for CBF12C. Genomic DNA was extracted from stem base samples and fragmented by ultrasonicator. DNA end-repair, A-tailing and adaptor ligation were performed according to Bartlett et al. (2017), using NEBNext Adaptor (New England Biolabs). HvNAC013 and CBF12C were produced as gluthation-S-transferase (GST) fusion proteins in Escherichia coli and co-incubated with DNA libraries as described in Bartlett et al. (2017) with minor modifications. Eluted DNA was PCR enriched using Phusion DNA polymerase (New England Biolabs) and NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, New England Biolabs). DAP-seq libraries were sequenced on NovaSeq 6000 using SP flowcell (Illumina) with 2x150 bp reads.