title: Low pass whole methylome enzymatic sequencing of four AML cell lines untreated and after treatment with different concentrations of 5-azacytidine (AZA), 5-aza-2′-deoxycytidine (DEC), Cytarabine (ARAC) and GSK3685032 (GSK5032) for 3 and 7 days
design: Low pass whole methylome enzymatic sequencing of four AML cell lines untreated and after treatment with different concentrations of 5-azacytidine (AZA), 5-aza-2′-deoxycytidine (DEC), Cytarabine (ARAC) and GSK3685032 (GSK5032) for 3 and 7 days
library_construction_protocol: Commercially available AML cell lines MOLM-13, Kasumi-1, KG-1a and THP-1 were used.  AML cell lines MOLM-13, Kasumi-1, KG-1a and THP-1 were cultured in American Type Culture Collection (ATCC)-modified RPMI 1640 medium (Thermo Fisher Scientific, A10491), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, 10500) and 1% penicillin-streptomycin (100 U/mL penicillin, 100 μg/mL streptomycin) (Thermo Fisher Scientific, 15140). All cells were kept in a humidified incubator at 37°C with 5% CO2, cells were passaged every two or three days and checked routinely for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, LT07-318). 5-azacytidine (AZA; Sigma-Aldrich, A1287), 5-aza-2′-deoxycytidine (DAC; Sigma-Aldrich, A3656), GSK-3685032 (GSK5032; MedChemExpress, HY-139664) and Venetoclax (VEN; MedChemExpress, HY-15531) stock solutions were dissolved in Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, D8418), while cytarabine (Sigma-Aldrich, C1768) was dissolved in distilled H2O (dH2O) to a final concentration of 10 mM. Stocks were kept at -20°C for up to one month, while cytarabine was stored at 4°C. 1.5*105 cells in 500ul of RPMI medium were treated for 24h (VEN), 3 days (AZA, DAC, GSK5032 and ARAC) and 7 days (GSK5032). Untreated controls were incubated with equivalent concentrations of DMSO or dH2O. DNA was extracted using Quick-DNA/RNA Kit (Zymo Research, D7001 and D7003). The manufacturer's instructions were followed with the addition of in-column treatment with RNase A/T1 (Thermo Fisher Scientific, EN0551). After binding of DNA to the extraction columns, DNA was incubated with 4U RNase A/T1 for 15 minutes at room temperature. DNA was quantified using a NanoDrop 2000 spectrophotometer.  200 ng DNA was diluted in 0.1X TE with 0.001 ng methylated pUC19 control DNA and 0.02 ng unmethylated lambda control DNA to a total volume of 50 µl. DNA was fragmented by sonication (Covaris S220) to an average fragment length of 302 bp. Sequencing libraries were prepared using the NEBNext Enzymatic Methyl-seq Kit (New England Biolabs, E71207S/L) following the manufacturer's instructions for standard library inserts. In short, after end-prep of the DNA, sequencing adaptors were ligated to the fragmented DNA. Following, methylated and hydroxy-methylated cytosines were oxidized by TET2 and the DNA was denatured by incubation with formamide at 85°C for 10 minutes. Cytosines were deaminated by the APOBEC enzyme and libraries were PCR-amplified for 4 cycles. Sequencing libraries were quantified using the Qubit 1X dsDNA High Sensitivity assay (Thermo Fisher Scientific, Q33231), and library size was estimated on a Fragment Analyzer or 4150 TapeStation with the DNF-474 HS NGS Fragment Kit or D1000 kit (Agilent), respectively.