Instrument: NextSeq 500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: Genomic DNA was extracted following procedure described in Thomas et al., 1993 (Thomas MR, Scott NS. 1993. Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs). Theoretical and Applied Genetics. 86(8):985-90. doi: 10.1007/BF00211051. PMID: 24194007). DAP-seq experiment was performed as described in Bartlett et al., 2017 (Bartlett, A., O'Malley, RC., Huang, SC. et al. Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat Protoc 12:1659-1672 (2017). https://doi.org/10.1038/nprot.2017.055). The DAP-Seq DNA library is prepared from fragmented naked genomic DNA ligated with a short DNA sequence (20bp) encoding a truncated Illumina TruSeq adapter. Separately, a construct containing a HALO-tagged transcription factor is translated by in vitro (rabbit reticulocyte) expression system. The affinity-tagged protein is immobilized on the appropriate affinity resin. The DNA affinity purification step begins by combining the DNA library with the immobilized transcription factor. After the wash steps remove the unbound DNA, the bound DNA fraction is eluted. The eluted DNA fragments are PCR amplified with a full-length indexed TruSeq primer and directly sequenced on the Illumina NextSeq 500.