GSM9135009: MDSL Cell line DB2115-treated 8h RNA-seq Rep3; Homo sapiens; RNA-Seq
Human bone marrow aspirates were obtained from in accordance with the Italian laws and the policies and practices of the Ethical Committee of Humanitas Research Hospital. Patients were classified following the updated WHO classification of tumors and lymphoid tissues guidelines 1 and stratified by risk of evolution following the IPSS-R parameters. Bone marrow mononuclear cells (BMNCs) were isolated by density gradient separation with Lympholyte®-H Cell Separation Media (Cedarlane corporation, Burlington, USA) according to manufacturer's instructions, frozen in fetal bovine serum (FBS) (Lonza, Basel, Switzerland) containing 10% DimethylSulfoxide (DMSO) (Sigma Aldrich, St. Louis, Missouri, USA) and cryopreserved in liquid nitrogen. Bone marrow HSPCs were isolated based on CD34+ surface marker using the MACS CD34 microbead kit on autoMACS (Miltenyi Biotech Inc, San Diego, CA). MDS-L cells were kindly provided by Dr. Daniel Starczynowski, Cincinnati Children's Hospital Medical Center and were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% P/S and 10ng/mL of hrIL-3 (PeproTech, Paisley, UK). Cells were grown at 37°C and 5%CO2 and treated with 3uM DB2115 (Cat. No. HY-124676A Medchem, NJ, USA) for the specified time. Libraries for CD34+ mRNA sequencing were prepared by using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara). Libraries for MDS-L mRNA sequencing were prepared by using the Illumina Stranded mRNA Prep (Illumina). All samples were sequenced at an average of 40 million 50-bp pair-end reads with Novaseq 6000. All RNA-seq data sets were processed in paired-end.