Cell culture, CRISPR-Cas9 gene editing, and generation of stable cell lines

C3H10T1/2 mouse mesenchymal stem cells (ATCC) were cultured in DMEM with 10% fetal bovine serum (FBS) and supplemented with 1% GlutaMax. Drosophila S2 cells were cultured in Schneider’s Drosophila medium (ThermoFisher) containing 10% heat-inactivated FBS. All cell lines tested negative for mycoplasma contamination. To generate knockout cell lines, 3.5 × 10⁵ C3H10T1/2 cells were nucleofected with ribonucleoprotein (RNP)-mediated CRISPR-Cas9 using the Alt-R CRISPR-Cas9 System (IDT) and Lonza Amaxa® SE Cell Line 4D Nucleofector kit (V4XC-1032, program CA-133). Synthetic crRNA guides were designed (Additional File 2: Table S1) and combined with Alt-R® CRISPR-Cas9 tracrRNA, ATTO 550 (IDT) and coupled to Alt-R® S.p. Cas9 Nuclease V3 following the IDT protocol “Delivery of ribonucleoprotein complexes using the Lonza® Nucleofector System” prior to nucleofection. The transfected cells were incubated for 48 h. Single ATTO 550-positive cells were then sorted into 96-well plates. Individual clones were then expanded and validated by MiSeq sequencing using specific primers for target loci (Additional File 2: Table S1). Three biological clones were chosen and expanded for each knockout cell line and used in all subsequent assays. C3H10T1/2 cells overexpressing the H3K36M mutation were provided by the lab of Dr. Chao Lu (Stanford University), and C3H10T1/2 NSD1/2-DKO and C3H10T1/2 NSD1/2-SETD2-TKO cells were provided by the lab of Dr. C. David Allis (Rockefeller University).