Homo sapiens
Protocols: 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*105 cells per ml were treated for three or seven days with treatment being refreshed every day. RNA 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) and DNase I (Zymo Research, E1010). After binding of RNA to the extraction column, RNA was treated with 5U DNase for 15 minutes at room temperature. RNA was quantified using a NanoDrop 2000 spectrophotometer.  Directional total RNA sequencing libraries were prepared from 500 ng RNA using the NEBNext rRNA Depletion Kit v2 (New England Biolabs, E7405L), NEBNext Ultra™ II Directional RNA Library Prep (New England Biolabs, E7765S) and NEBNext Multiplex Oligos for Illumina (New England Biolabs, E7335S) according to the manufacturer's instructions with slight adjustments as follows. After removal of ribosomal RNA using DNA probes and digestion with RNase H and DNase I, RNA was fragmented for 10 minutes at 94°C for an average library insert size of 300 nucleotides. First strand cDNA synthesis was performed for 10 minutes at 25°C, 50 minutes at 42°C and 15 minutes at 70°C. Libraries were PCR amplified for 9 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).
isolate
not applicable
disease
adult T acute lymphoblastic leukemia
organism
Homo sapiens
organism
Homo sapiens
cell_line
Loucy
collection date
not collected
scientific_name
Homo sapiens
common name
human
organism part
immune system
cell_type
T lymphoblast
developmental stage
adult
geographic location (country and/or sea)
not collected