GSM2870527: A.pp 4074 NarP DAP-Seq; Actinobacillus pleuropneumoniae; OTHER
The genomic DNA of A. pleuropneumoniae was prepared using a DNeasy Blood & Tissue Kit (Qiagen). 1 μg genomic DNA was sheared to an average length of 300-500 bp by ultra-sonication for 16 min at 20% amplitude with 2 seconds on and 8 seconds off on ice. The size of the sheared genomic DNA was checked using 1 % agarose gel. The sheared DNA fragments were treated with Klenow enzyme to generate blunt ends, phosphorylated and 3'-end adenylated followed by ligation with paired-end adapters. The fragments with adapters on both ends were enriched by a ten-cycle PCR and purified using a QIAquick PCR Purification Kit. The adapter-containing DNA fragments were then subject to DNA affinity purification. Briefly, the DNA fragments were mixed with the purified his-tagged NarP in the reaction buffer (10 mM Tris HCl, pH 7.5, 1 mM DTT, 5 mM MgCl2, 50 mM acetyl phosphate and 25% glycerol) in which acetyl phosphate was used to activate NarP. The mixture was incubated at 25 °C for 30 minutes and then incubated with 30 μl of Ni-NTA resin that had been equilibrated in binding buffer 2 (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 50 mM KCl, and 25% glycerol) for 30 min. The resin was washed three times with binding buffer 2 and the protein was eluted with elution buffer (20 mM sodium phosphate buffer pH 8, 500 mM NaCl, and 500 mM imidazole). The DNA bound to NarP was recycled using a Qiaquick PCR purification kit.
GEO Accession
GSM2870527