GSM8026609: Human_CTCF-MBD2-random_CAP-Methyl-SELEX_cycle3_rep2; Escherichia coli; SELEX
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 CAP-SELEX experiments were carried out using a previously established protocol. Briefly, dilute 10–200 ng of purified Flag and SBP-tagged proteins into a 25 µl volume of binding  buffer (140 mM KCl, 5 mM NaCl, 2 mM MgSO4, 3 μM ZnSO4, 100 μM EGTA, 1 mM K2HPO4, in a solution containing approximately 10 μmol DNA select ligand in 20mM HEPES, pH 7.0, and incubate at room temperature for 20 minutes. Subsequently, add two volumes of 0.2% BSA, 0.1% Tween 20 in binding buffer to the preblocked streptavidin-coated magnetic Sepharose beads (1.25 μl; GE Healthcare Streptavidin Mag Sepharose) and the mixture was incubated with strong  medium for 2 h at room temperature. The beads were washed 5 times with binding buffer. Protein_x0002_DNA complexes were eluted from the beads using 50 μl of 10 mM biotin (Sigma) in binding buffer. Transfer the eluate to M2 containing 50 μl of binding buffer. Add 1.25 μl anti-Flag magnetic beads (Sigma) to a fresh plate and shake at 800 r.p.m. Leave at room temperature for 20 minutes. The beads are washed ten times with binding buffer and suspended in 0.1% Tween 20, 0.5mM EDTA, 10mM Tris-Cl, pH 8.0, and transferred to a PCR plate. DNA was then eluted from the beads by incubation at 95 °C for 10 min. A 10 μl aliquot of the bead suspension was transferred to a new PCR plate and amplified by PCR Amplify DNA. The amplified selected ligands are then subjected to sequencing and new cycles of CAP-SELEX (up to 3 cycles in total). After each cycle, purified PCR products were pooled and sequenced using an Illumina HiSeq Xten sequencer.