In vitro gennomic DNA binding assay and sequencing using recombinant HY5 protein.. Arabidopsis thaliana

Genomic DNA (gDNA) was extracted from the whole aerial portion of 2-4-week-old Arabidopsis Col-0 plants using a DNeasy Plant Midi Kit (Qiagen) and sheared into lengths of approximately 150-200 nt by Covaris S (Covaris). Approximately 10 µg of recombinant HY5-HA protein were mixed with a 60 µl volume of Dynabeads Protein G (VERITAS) and 5 µg of anti-HA antibody (abcam) in 400 µl of solution A (1×PBS pH7.4, 5 mM EDTA, 0.05 % TritonX100, 5 % glycerol) and then incubated with constant rotation for 2 hours at room temperature. The beads combined with HY5-HA were washed twice using solution A and resuspended in 500 µl of solution A. The sheared gDNA fragments (25 µg) were added into the solution containing the beads with HY5-HA and incubated with constant rotation overnight at 4◦C. The HY5-HA beads bound with gDNA fragments were washed four times in solution A and resuspended in 100 µl of solution B (10 mM Tris-HCl pH8.0, 10 mM EDTA, 0.5 % SDS). This was mixed with 5 µl of proteinase K (2 mg/ml) and incubated for 2 hours at 37◦C. The gDNA fragments were recovered by phenol:chloroform, and chloroform extractions followed by ethanol precipitation. A library for next-generation sequencing was constructed using the TruSeq ChIP Sample Prep Kit (Illumina) and the pair-end sequences were sequenced using a MiSeq sequencer (Illumina) according to the manufacturer’s instructions.