Kasumi 1 cell line

Informed consents were obtained in accordance with the Declaration of Helsinki. Samples were collected and analyzed under San Luigi Hospital internal institutional ethical committee–approved protocol (approval number 201/2014). Eighty-two BM and 8 PB specimens from AML patients at diagnosis, 15 PB from AML patients after therapy and 16 BM and 18 PB from healthy subjects were collected. All the patients have been previously characterized at the cytogenetic level by conventional karyotyping and screened by reverse transcriptase-PCR for the presence of the most frequent fusion transcripts. Mutations or internal tandem duplication of both Fms Related Tyrosine Kinase 3 (FLT3) and of Nucleophosmin 1 (NPM1) genes were also characterized. Acute promyelocytic leukemia samples were excluded from the study.
The human Kasumi-1 cell line was purchased from ATCC and cultured in RPMI-1640 supplemented with 20% fetal bovine serum (FBS), 500 U/mL penicillin, and 0.5 mg/mL streptomycin. Cells were cultured at 37 °C in a humidified atmosphere flushed with 5% CO₂.

CUT&RUN experiments were carried out as previously described (Skene et al., 2018 (link)) with the following modifications: 1–2.5×10⁵ cells were isolated by FACS as described in sections above, bound to Concanavalin A coated magnetic beads (Bangs Laboratories), and permeabilized with 0.025% (wt/vol) digitonin. Permeabilized cells were incubated overnight at 4°C with 5ug of anti-H3K27me3 (Active Motif) and then washed before incubating with protein A-MNase fusion protein (a gift from S. Henikoff) for 15 minutes at room temperature. After washing, cells were incubated in CaCl₂ to induce MNase cleavage activity for 30 minutes at 0°C. The reaction was stopped with 2xSTOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A, 50 μg/mL glycogen, and 2pg/mL of yeast spike-in DNA). Histone-DNA complexes were isolated from insoluble nuclear chromatin by centrifugation and DNA was extracted with a NucleoSpin PCR Clean-up kit (Macherey-Nagel). For CUT&RUN quantitative PCR, human Kasumi-1 cell line (ATCC CRL-2724) were added before binding the cells to Concanavalin A beads for internal standard instead of yeast spike-in DNA.