CRISPR-Cas9 Knockout Generation

HEK293 and CCD-841-CoN SALL2 total knockout clones were obtained by CRISPR-Cas9, as described in Escobar et al. (2015) and Hermosilla et al. (2018). The HEK293 SALL2 E1A-knockout cell model was obtained by electroporation at 1100 volts per 20 milliseconds (NEON Transfection System, Thermo Fisher Scientific), with a vector encoding Cas9, a specific SALL2 RNA guide (pX330, Addgene), and GFP or RFP-containing vector used as a transfection marker. Control cells were electroporated using the GFP or RFP-containing vector alone. Supplementary Table 1 indicates the specific human SALL2 guide RNAs and primers used for PCR reactions and sequencing. We collected 1000 cells by GFP sorting from which ten clones were obtained. From three clones, genomic DNA was purified and sequenced, giving one clone with the expected deletion (clone 17). Validation of SALL2 E1A included Sanger sequencing (performed at Pontificia Universidad Católica Sequencing Facility, Santiago, Chile) and Western Blot analysis (Supplementary Figures 2, 3).