Haploid Cell Line CRISPR Editing

Baz2B-KO (HZGHC000523c005, 16 bp deletion in the coding exon in Baz2B was edited by CRISPR-Cas9) and its parental Hap1 cell lines were purchased from Horizon Discovery (Cambridge, UK). Cells were cultured in IMDM (from Gibco™, serum-free medium) supplemented with l-glutamine, phenol red indicator, and 10% FCS. Cells were typically maintained at 40–90% confluence and passaged every 2–3 days. The two cell lines were treated separately.
An inducible U2OS cell line expressing BAZ2B-halo was generated by cloning the BAZ2B gene (UniProt: Q9UIF8, codon optimised by Genescript) into the pEBTet GW_Halo plasmid vector, transfecting and selecting plasmid-containing cells in McCoys 5A medium (ThermoFisher #16600082) with 10% FBS (BioSELL, #S0615) supplemented with 1 mM sodium pyruvate (Sigma, #S8636), 1% penicillin-streptomycin (Sigma #P0781) and 1 μg/ml puromycin (Sigma, #P9620) in a humidified 5% CO2 incubator at 37°C for 1–2 weeks (64 (link),65 (link)). For imaging, the cells were grown in μ-Slide 8-well glass bottom slides (Ibidi) for 48 h and gene expression was induced with 0.1 μg/ml doxycycline for a further 24 h. The BAZ2B-halo was stained by incubation with 100 nM JF646-halo for 1 h and the cells were fixed with 4% PFA.