Illumina MiSeq sequencing; HT-SELEX of recombinant C2H2 zinc finger domain of Sall4
HT-SELEX of recombinant C2H2 zinc finger domain of Sall4
To generate SELEX libraries, two overlapping single-stranded oligonucleotides (ordered from Integrated DNA Technologies) were annealed and filled-in using Klenow polymerase (NEB ref. M0210S). Double-stranded DNA libraries were purified using the QIAquick PCR Purification Kit (Qiagen ref. 28104) and controlled on a 10% polyacrylamide gel. To perform SELEX, SELEX libraries (1.5µg for the first cycle, 200ng for subsequent cycles) were mixed with 1µg of hexahistidine-tagged SALL4 ZFC4 (purified from E.coli) in 100µl of SELEX buffer (50mM NaCl, 1mM MgCl2, 0.5mM EDTA, 10mM Tris-HCl pH7.5, 4% glycerol) freshly supplemented with 5µg/ml Poly(dI-dC) and 0.5mM DTT. Following a 10min incubation at room temperature on a rotating wheel, 50µl of Ni Sepharose 6 Fast Flow beads (GE Healthcare), previously equilibrated in SELEX buffer, was added into each sample and incubated for an additional 20min at room temperature on a rotating wheel. To remove non-specifically bound oligonucleotides, beads were washed 5 times with 1ml of SELEX buffer, freshly supplemented with 0.5mM DTT. Between each wash, samples were incubated for 5min at room temperature on a rotating wheel and centrifuged for 1min at 2,000 rpm. After the final wash, beads were resuspended in 100µl H2O and used directly for PCR using the high-fidelity Phusion DNA polymerase (NEB ref. M0530S). The minimum amount of PCR cycles required to amplify each library was determined by running samples amplified with increasing PCR cycle numbers on 10% polyacrylamide gels. Amplified libraries were purified using the QIAquick PCR Purification Kit and used directly for sequencing, or used for subsequent rounds of SELEX.