title: GSM5958395: bZIP44_Col-B_p1; Arabidopsis thaliana; OTHER
library_construction_protocol: Genomic DNA extracted method and DAP-seq experiment was performed as described in Li et al., 2022 (Li M., Huang SS.C. DNA Affinity Purification Sequencing (DAP-Seq) for Mapping Genome-Wide Transcription Factor Binding Sites in Plants. In: Bilichak A., Laurie J.D. (eds) Accelerated Breeding of Cereal Crops. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1526-3_15) and Bartlett et al., 2017 (Bartlett, A., O'Malley, RC., Huang, SC. et al. Mapping genome-wide transcription-factorbinding sites using DAP-seq. Nat Protoc 12:1659-1672 (2017). https://doi.org/10.1038/nprot.2017.055).  The DAP-seq DNA library was prepared from fragmented genomic DNA ligated with a short DNA sequence (20bp) encoding a truncated Illumina TruSeq adapter.  In DAP-seq, a protein expression construct containing HALO-tagged transcription factors are translated by in vitro (rabbit reticulocyte) expression system in single DAP experiment to form homodimers. In double DAP-seq, one construct containing Halo-tagged transcription factor and one containing SBP-tagged transcription factor were co-expressed by in vitro  (rabbit reticulocyte) expression system to form heterodimers. The affinity-tagged proteins are immobilized on the Halo ligand magnetic beads.  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 DNA fragments bound to in vitro expressed proteins (homodimers or heterodimers) were eluted from affinity beads, PCR amplified with a full-length indexed TruSeq primer and directly sequenced on Illumina NextSeq 500.