title: GSM9327959: MDSL Cell line PU.1 knock out Rep3; Homo sapiens; RNA-Seq
library_construction_protocol: 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.   The crRNAs for PU.1 knock-out were designed using Benchling tool (crRNA sequences are listed in Supplemental Methods). Custom designed crRNAs and Alt-R™ CRISPR-Cas9 tracrRNA 20nmol (Cat. No. 1072533, IDT) were mixed in Nuclease-free Duplex Buffer (IDT) at a molar ratio of 1:1, annealed at 95˚C for 5 minutes and cooled down to room temperature (ramp scale -0.1˚C/sec). The annealed sgRNAs were incubated with Cas9 TrueCut V2 (Cat. No. 36499, Thermo Fisher) in a molar ratio of 1:1 at room temperature for 15 minutes before transfection. After complex formation, 1×106 MDS-L cells were resuspended in P3 Buffer and 10.8 µM Electroporation Enhancer and then transfected using P3 primary cell Nucleofector kit (Cat. No. V4XP3024, Lonza) with Amaxa Nucleofector device (Lonza). 48 hours after electroporation, cells were harvested for PU.1 knock-out validation by Western Blot, and for RNA extraction and library preparation. Libraries for CD34+ mRNA sequencing were prepared by using the SMART-Seq v4 Ultra Low Input RNA Kit (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.