PbAP2-TR plays a role in regulating major transcriptomic changes from early to late trophozoite [PbAP2-TR_DIP-seq]
To investigate the DNA-binding property of AP2 domains of PbAP2-TR, DNA immunoprecipitation followed by high-throughput sequencing (DIP-seq) analysis was performed. Recombinant AP2 domains 1–2 or AP2 domain 3 fused with maltose-binding protein (MBP) were mixed with the P. berghei genomic DNA fragmented via sonication, and protein-DNA complex was harvested using amylose resin. The obtained DNA fragments were sequenced via the next generation sequencing. Overall design: DNA fragments encoding the AP2 domains 1–2 or AP2 domain 3 of PbAP2-TR were each cloned into the expression vector pMal-c5X (NEB). Escherichia coli strain DH5a transformed with these plasmids was cultured for 12 h at 37 °C. Next, expression of the MBP-fused protein was induced by adding isopropyl ß-D-thiogalactopyranoside (final concentration of 200 nM) in the culture and incubating for 9 h at 25 °C. Recombinant AP2 domains fused with MBP was purified using amylose resin, and eluted with 10 mM maltose solution. Subsequently, the MBP-fused homeodomain was incubated with P. berghei ANKA genomic DNA fragments in binding/washing buffer (10 µM ZnSO4, 2 mM MgCl2, 2 mM Tris-HCl at pH 7.4, 100 mM KCl, and 10% glycerol) for 30 min. The protein/DNA solution was further mixed with amylose resin and incubated for 30 min. After incubation, the resin was washed three times with binding/washing buffer, and bound protein-DNA complexes were eluted with 10 mM maltose solution. A sequencing library was prepared from the DNA fragments and sequenced using Illumina NextSeq. Before their use for DIP, genomic DNA fragments were also sequenced as an input, the raw data of which have been already deposited on Gene Expression Omnibus under the accession number GSE159045.