Hes1 knockdown and overexpression.
In brief, K562 cells were nucleofected with Hes1 overexpression and knockdown plasmids on Amaxa 4d nucleofector from Lonza and incubated for 3 days. Hes1 was overexpressed by miGR1-Hes1 plasmid. Hes1 knockdown was performed by shRNA against Hes1 open-reading frame. ShRNA-Hes1 plasmids were a kind gift from Dr Adolo Ferrando, Columbia University. MigR1 control and MigR1-Hes1 plasmids were a kind gift from Avinash Bhandoola lab (NIH, USA).
Generation of CRISPR-based knockout system for MIR182 locus.
CRISPR/Cas9 system has been used to modify genomic loci of clinical importance. A stepwise process to delete the MIR182 locus was undertaken starting with HEK293 to standardise knockout method (Supplementary Figure 5a). To reduce the incidence of offtarget effects, three CRISPR plasmids (targeting 129409678, 129410425 and 129410482 according to GRCh37.p13 of human genome assembly) were nucleofected in K562 cell line (Figure 3c).