ZBTB10 binds the telomeric variant repeat TTGGGG and interacts with TRF2 [DNA-Seq validation of KO]

In order to investigate changes upon ZBTB10 depletion, we used CRISPR/Cas9 to induce a ZBTB10 knockout in HEK293 and U2OS cells. ZBTB10 knockouts were generated using three different guide RNAs targeting the second exon of the ZBTB10 gene. HEK293 and U2OS cells were selected in 750 ng/mL puromycin after transfection and gDNA was extracted. A circa 1100 bp long target sequence around the cut locus was amplified by PCR and Cas9 activity was tested in a T7 endonuclease assay. After confirming Cas9 activity, single cell sorting was performed and cells were expanded to screen for ZBTB10 knockout clones by Western blot using an anti-ZBTB10 antibody (Supplementary Table 1).