title: DAPseq of Sorghum bicolor: SbBES1 rep1
design: Protein preparation. The SbBES1-pHALO recombinant plasmid and an empty pIX-HALO plasmid (generating only the HALO peptide when translated) were in vitro translated with TNT Coupled Reticulocyte Lysate System (Promega). Two replicates were performed for each plasmid. the pIX-HALO plasmid was used as the input control. DAP-seq library preparation. Washed Halo-Tag beads were mixed with the in vitro translated proteins to pull down the Halo-Tag fused SbBES1 protein. Each pulled-down protein reaction was mixed with 500ng genomic DNA libraries for SbBES1 binding. The bound DNA was washed by PBST for 6 times, then released from Halo-Tag beads by denaturation at high temperature (98 C). The eluted DNA was enriched by PCR amplification to generate the sequencing libraries. The enriched DAP-seq libraries were purified and washed with AMPure XP beads and 80% ethanol for removing indexes. Resuspended DAP-seq libraries were quantified by Qubit and were confirmed by bioanalyzer analysis. DAP-seq library sequencing. DAP-seq libraries were sequenced on an Illumina NextSeq 550 (sequencing of libraries was set at 40 million and 1x75bp single-end reads). As a negative control, the input (pIX-HALO expression vector without any insertion of opening reading frame) was accounted for possible non-specific DNA binding in the reaction mixture, and copy number variations at specific genomic loci.