Supplementary Figure 4: Characterization of CRISPR–Cas9-generated HS-mutant MLEC lines.

The Ndst1^f/f MLECs were transiently cotransfected with plasmids encoding Cas9 and gRNA targeting Glce, Hs3st1, or Hs3st4 to derive Glce^−/− (a), Hs3st1^{–/ –} (b), and Hs3st4^−/− (c) cell lines, respectively. The Hs3st1^−/− cell line was transiently cotransfected with plasmids expressing Cas9 and gRNA targeting Hs3st4 to derive the Hs3st1^−/−;Hs3st4^−/− cell line (c). After puromycin selection, the transfected cells were cloned and screened by enzyme mismatch assay for gRNA-induced indel mutation. The identified indel mutations were further characterized by determination of the mutant nucleic acid sequences. Because of induced deletion larger than could be amplified by the PCR primers that amplify the gRNA-targeted region in the wild-type control, no gene sequencing data were obtained for Hs3st4 indels in the Hs3st4^−/− and Hs3st1^−/−;Hs3st4^−/− MLEC lines. Representative results are shown as the mean ± s.e.m. of 3 independent PCR analyses.