title: GSM5671175: Mutant CTCF construct R567W rep1; synthetic construct; OTHER
library_construction_protocol: Recombinant protein expression protocol: We used the NEB PURExpress system to produce N-terminal HALO tagged CTCF constructs and noticed better success rates than previous 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).