Generation of HUDEP-2 knockin cell lines

Cas9 single gRNA (sgRNA) RNPs were generated by mixing 0.5 µL of 40 µM Cas9 protein and 1 µL of 50 µM sgRNA (Synthego, 5′-aaa​caa​cUc​aga​ggg​UUc​cc-3′) at room temperature for 10 minutes. The RNP cocktail was then mixed with 5 µg single-strand oligodeoxynucleotides and 200 ng pMaxGFP, added to 2 × 10⁵ HUDEP-2 cells, and subjected to nucleofection with the Neon transfection system (Invitrogen, 1150 V, 30 ms, 2 pulses). After 1 week of cell culture, single GFP⁺ cells were sorted into individual wells of 96-well U-bottom plates with a BD Bioscience Aria cell sorter. After 2 weeks of clonal expansion, targeted deep sequencing was performed to identify clones with accurate homozygous deletion of CTCF-binding motifs. Two clones were selected for further experiments.