Stable cell line generation by random integration

To generate cell lines expressing H2B-HaloTag the corresponding cDNA was cloned into an expression plasmid driven by the CAG promoter that also contained an internal ribosome entry site that allowed co-expression of the G418 resistance gene (primers are listed in Supplementary Table ¹). For HaloTag-3xNLS expression, the pHTN CMV-neo vector (Promega), modified with 3x SV-40 NLS at the C-terminus (gift, James Rhodes), was used. In order to randomly integrate this expression cassette, ESCs in one well of a 6-well plate were transfected with 10 μg of expression vector using Lipofectamine 3000 (ThermoFisher) according to the manufacturer’s instructions. The following morning, transfected cells were passaged onto new plates at a range of cell densities. 5 h later, 400 μg/ml of G418 was applied. Cells were grown in the presence of G418 to select for stable integration events and approximately one week later resistant colonies were picked and expanded on 96-well plates. 96 well plates of putative positive clones were labelled with 500 nM Halo-TMR dye (Promega) for 15 min at 37 °C, washed in label free medium three times, and then incubated in medium without dye for 30 min at 37 °C. Individual clones with stable HaloTag fusion protein expression were identified by fluorescence microscopy.