GSM5708116: Truncated ZNF10_F1-F8 construct replicate 1; synthetic construct; OTHER
[Recombinant protein expression protocol] We used the NEB PURExpress or E. coli_BL21 system to produce N-terminal HALO tagged ZFP constructs and noticed better success rates than previously used SUMO-tagged constructs. [EMSA protocol] 20uL binding reactions were loaded onto 10% Tris-glycine gel in cold room and run for 50mins at 200V. [Sequencing sample prep protocol] The bound and unbound fractions of each sample were cut separately as thin gel slice, extracted by acrylamide gel extraction buffer _soaking a 55C for more than 30mins. Each fraction of DNA pool was purified and further amplified by uniquely indexed Illumina primers set for at least 12 cycles _annealing temperature 52C, extension time 30s. All samples were pooled together for Illumina Nextseq _1X 75bp single end run.