Illumina NovaSeq 6000 paired end sequencing; RNA-seq of MOLM-13 acute myeloid leukemia cell line treated with single and combination 5-Azacytidine and CB-5083
RNA-seq of MOLM-13 acute myeloid leukemia cell line treated with single and combination 5-Azacytidine and CB-5083
MOLM-13 cells were detached from by addition of trypsin. Cells suspensions were quenched with medium and centrifuged at 400 x g for 5 minutes to pellet for subsequent extraction. MOLM-13 cells were cultured in RPMI1640 supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin. Cells were treated with 450 nM CB-5083 (Cayman Chemical 19311) for 48 hours Total RNA was extracted using Trizol (Invitrogen) and purified using Direct-zol RNA Purification Kits (Zymo Research). Library construction was performed by Novogene UK (Cambridge, UK). A minimum of 1 ug (20 ng/Î¼l) of high-quality total RNA (extracted using Trizol, Invitrogen, and purified using Direct-zol RNA Purification Kits, Zymo Research) was supplied for sequencing. Novogene UK constructed cDNA libraries by purification of messenger RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification.