Cell lines and template reference materials

OCI-AML3 cells (DSMZ) and KG1a cells (ATCC) were cultured in 20% FB Essence (VWR Life Science) in RPMI media (Gibco). Genomic DNA was extracted from cell line lysates using Qiagen Gentra PureGene Cell Kit (Cat No./ID: 158745), according to the manufacturer’s protocol. Reference sequence plasmids for NPM1 alleles were lab-designed and purchased from Life Technologies. Single-zygosity plasmids (homozygous mutant or homozygous wild-type) contained one copy of the amplicon region per plasmid. Heterozygous plasmids contained one copy of wild-type and one copy of mutant amplicon region inserted in series. All plasmids were sequence-validated by Life Technologies and in house. OCI-AML3 cells and all mutation-containing plasmids had the “Type A” NPM1 insertion, which is the TCTG tetranucleotide duplication in position c.860_863dupTCTG ({"type":"entrez-nucleotide","attrs":{"text":"NM_002520.6","term_id":"262331543","term_text":"NM_002520.6"}}NM_002520.6), the sequence of the insertion that is present in ~80% of AML patients positive for the NPM1 insertion.