GSM6632379: CTCF-DBD_oligoLib1_Methyl-SELEX_cycle4_rep1; Escherichia coli; OTHER
The pellet was suspended in 10 ml buffer A (0.5 mg/ml Lysozyme, 50 mM tris-HCl pH 7.5, 300 mM NaCl, 10 mM Imidazole, 1 mM DTT, 1 mM PMSF) and lysed by sonication at ten seconds interval for 10 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 beads was set at 4 ℃ for 30min before being eluted with Buffer A containing 300 mM imidazole The HT-SELEX experiments were carried out using a previously established protocol12. Briefly, selection ligands consisted of 41 bp (with a 19 bp core CTCF motif sequence flanked by 11 bp random sequences on each side) adapted to Illumina HiSeq Xten sequencing system with multiplexing indices. Ligand libraries were then generated by PCR amplification using primers in Extended Data Table 6 and 41-bp SELEX ligands as templates. The input library was amplified for 16 cycles using Q5® High-Fidelity DNA Polymerase (NEB). All PCR reactions were performed according to the manufacturer's manual. Libraries were sequenced using an Illumina HiSeq Xten sequencer to analyze the identity of each base (A, T, C, G). Then, 150 ng protein and 5 μL of selection ligand were added to Promega binding buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM MgCl2, 4% glycerol, 0.5 mM EDTA, 5 μg/ml poly -dIdC (Sigma P4929)) until the total volume reaches 25 μL. After a 30-minute incubation at room temperature, 150-µL Promega buffer (without poly-dIdC) containing 10-µL Nickel Sepharose 6 Fast Flow resin (GE) equilibrated in binding buffer was added to the mixture, followed by incubation for 60 minutes with gentle shaking. After binding, the Nickel Sepharose beads were washed for 12 consecutive times with 200 μl Promega Binding Buffer (without poly-dIdC) with gentle shaking. Each wash took 5 min. Subsequently, the residual water was carefully removed from the beads by centrifugation at 500×g for 30 seconds. Then, beads with bound DNA were resuspended in 200 μL of mili-Q water. Finally, approximately 20 μL of beads was PCR amplified for 18 cycles using Phusion DNA polymerase (NEB), and the resulting PCR product was used as the selection ligand for the next cycle of SELEX. This process was repeated four times. After each cycle, purified PCR products were pooled and sequenced using an Illumina HiSeq Xten sequencer.The methylation sensitive-SELEX experiments were carried out using a previously established protocol13. CpG methylation is modified by the CpG-specific methyltransferase M.SssI. The methylation protocol was adapted from13 as follows: 2.5 μL (10U, for initial library) or 1.25 μL (5U, for cycles 1 to 3) CpG methyltransferase M.SssI (NEB; twofold excess units in input and SELEX cycle beyond), with 0.4 μL (for initial library) or 0.2 μL (for cycles 1 to 3) S-adenosylmethionine (SAM), 3.4 μL of 50 mM MgCl2, and 0.2 μL of 100 mM DTT to DNA ligand in a total volume of 20 μL. The mixture was incubated at 37°C for 3 hours to methylate CpG dinucleotides in double-stranded DNA ligands, then at 65°C for 20 minutes to inactivate the M.SssI enzyme before each selection cycle. The methylation status of the ligands was examined by the methylation-specific restriction enzyme BstBI digestion (Extended Data Fig. 5a). CpG methylated DNA ligands and unmethylated DNA ligands SELEX assays were performed in parallel for each protein in separate tubes, and the selection process was repeated for up to 4 cycles to enrich for binding sequences. After each cycle, purified PCR products from each cycle of methyl-SELEX and HT-SELEX were pooled separately and sequenced using an Illumina HiSeq Xten sequencer.