Homo sapiens
Protocols: Commercially available T-ALL cell lines; LOUCY, JURKAT, MOLT-4, MOLT-3, DND-41, TALL-1, SUPT-1, CCRF-CEM, PEER, ALL-SIL, HPB-ALL were used.    T-ALL cell lines (LOUCY, JURKAT, MOLT-4, MOLT-3, DND-41, TALL-1, SUPT-1, CCRF-CEM, PEER, ALL-SIL, HPB-ALL) were cultured in American Type Culture Collection- RPMI 1640 medium (Gibco Thermo Fisher Scientific, A10491). Cell culture medium was supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco Thermo Fisher Scientific, 10500) and 1% penicillin-streptomycin (100 U/mL penicillin, 100 μg/mL streptomycin) (Gibco Thermo Fisher Scientific, 15140). All cells were kept in a humidified incubator at 37°C with 5% CO2, passaged every second or third day and checked regularly for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, LT07-318).  For treated cell, 5-azacytidine (Sigma-Aldrich, A1287), 5-aza-2′-deoxycytidine (Sigma-Aldrich, A3656) and GSK-3685032 (MedChemExpress, HY-139664) were dissolved in DMSO (Sigma-Aldrich, D8418) to create a stock of 10 mM. Singel-use aliquots were kept at -20°C up to 1 month. 3*10^5 cells per ml were treated for three or seven days with treatment being refreshed every day. Untreated controls were incubated with equivalent concentrations of DMSO. 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 and 0.02 unmethylated lambda control DNA. DNA was fragmented by sonication (Bioruptor, Diagenode) to an average fragment length of 290 base pairs. Sequencing libraries were prepared using the NEBNext Enzymatic Methyl-seq Kit (New England Biolabs, E7120S and E7120L) following the manufacturer's instructions for standard library inserts. In short, after end-prep of the DNA, sequencing adapters were ligated to the fragmented DNA. Following, methylated and hydroxy-methylated cytosines were oxidized by TET2 after which 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 Bioanalyzer with the high sensitivity DNA assay (Agilent, 5067-4626).
disease
T-cell acute lymphoblastic leukemia
organism
Homo sapiens
organism
Homo sapiens
collection date
not collected
scientific_name
Homo sapiens
common name
human
organism part
immune system
developmental stage
child
genotype
N/A
isolate
not applicable
cell_line
DND-41
cell_type
T lymphoblast
geographic location (country and/or sea)
not collected