GSM4404218: PSPPH_0857_cycle2_Rep1; Pseudomonas savastanoi pv. phaseolicola 1448A; OTHER
The pellet was suspended in 12 ml buffer A and lysed by sonication at ten seconds interval for 20 minutes and then centrifuged at 4 °C (12000 rpm, 30 min) to collect protein supernatant. The supernatant was filtered with a 0.45 μm filter and the filtrate was added into a Ni-NTA column (Bio-Rad) which had been equilibrated with buffer A before using. The Ni-NTA column was eluted with 30 ml gradient from 60 to 500 mM imidazole prepared in buffer A gradually. HT-SELEX was performed by using selection ligands containing an 8 bp barcode before and after the 40 bp randomized region for Illumina system. The ligand libraries were produced by PCR amplification using the primers in Supplementary TableX and 40 bp randomized oligo as template. The libraries were sequenced using Illumina HiSeq Xten sequencer to analyze the uniformity of each base (A, T, C, G). Then, 100-200 ng protein and 5 ul selection ligands were added into Promega binding buffer (10mM Tris pH7.5, 50mM NaCl, 1 mM DTT, 1mM MgCl2, 4% glycerol, 0.5mM EDTA, 5µg/ml poly-dIdC (Sigma P4929)) until the total volume reached 25 ul. After incubating for 30 min at room temperature, the 150 ul Promega buffer (without poly-dIdC) containing 10 ul Ni Sepharose 6 Fast Flow resin (GE Healthcare 17-5318-01) equilibrated in binding buffer was added into the mixture and incubated for 60 min with a gentle shaking at room temperature. After binding, the resin beads were consecutively washed with a gentle shaking for 12 times with 200 μl of Promega binding buffer (without poly-dIdC). It took 5 min for each washing. Subsequently, the residual moisture on beads was carefully cleared by a soft centrifuging at 500 g for 30 s, and then the bound DNA was re-suspended by using 200 uL double-distilled water. Finally, about 20 uL bound DNA was performed for PCR amplification using Phusion DNA polymerase (NEB M0530L) with the primers in Supplementary TableX, and the resulting PCR products were used as selection ligands for the next cycle of HT-SELEX. This process was repeated four times. After each cycle, the purified PCR products from each HT-SELEX cycle were pooled and sequenced using Illumina HiSeq Xten sequencer.
GEO Accession
GSM4404218