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Creation of MCF10A isogenic cell lines with cohesin gene deletions.(A) Top, schematic diagram shows the deletion strategy for genes encoding cohesin subunits RAD21, SMC3, and STAG2 using two sgRNAs targeting the 5'UTR and the 3'UTR of each gene. Bottom, heterozygous clones were identified by PCR using specific primer pairs flanking the deletion region. Representative DNA gel shows the PCR products yielded using specific primer pairs for MCF10A parental and RAD21+/- deletion clone. M, ladder marker. (B,C,D) Schematic deletion strategy and summary of the allele sequences for the STAG2 homozygous deletion clone, and the RAD21 and SMC3 heterozygous deletion clones. (E) RNA levels of the targeted genes in MCF10A cohesin-deficient clones. (F) Representative immunoblot and (G) quantification of cohesin protein levels. γ-tubulin was used as loading control. n = 3 independent experiments, mean ±s.d., one-tailed student t test: **p≤0.01; ****p≤0.0001. Guide RNAs and PCR primers can be found in Supplementary file 1.
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