GSM8744476: 14M; dBM; NGS-3102; Homo sapiens; RNA-Seq
Flow cytometry analysis in whole blood and bone marrow samples: Peripheral blood (PB) and BM samples were collected into tubes containing EDTA, and mononuclear cells (PBMCs and BMMCs) were isolated over a density gradient using Ficoll-Paque Plus (GE Healthcare) within 24 hours of sample collection. Following density gradient centrifugation, at least one million single cells per sample were stained with fluorochrome-coupled antibodies. The monoclonal antibodies used for labelling of samples were: ECD-labelled anti-HLA-DR (Immu-357), PC7-labeled anti-CD33 (D3HL60.251), APC-labeled anti-CD14 (RM052), FITC-labeled anti-CD11b (94), PE-labeled anti-CD15 (80H5), all antibodies from Beckman Coulter-Immunotech. Total MDSCs, monocytic MDSCs (M-MDSCs) and granulocytic myeloid derived suppressor cells (PMN-MDSCs) were measured by flow cytometry using the NAVIOS cytometry system (Beckmann Coulter) and analysed using KALUZA software (Beckmann Coulter).  Gating strategy is shown in Figure 1. Briefly, total MDSCs were assessed as CD11b+CD33+ HLADRlow/−, while M-MDSCs were gated as CD11b+CD33+ HLADRlow/−CD14+ CD15− and PMN-MDSCs as CD11b+ CD33+ HLA-DRlow/−CD14− CD15+. Cell sorting of MDSCs was performed under aseptic conditions using 70-micron nozzle with a BD FACS Aria III (BD Biosciences). RNA Isolation and 3′ RNA sequencing. RNA from FACS-sorted M-MDSCs was isolated using Nucleospin RNA XS kit (Macherey-Nagel), according to the manufacturer's instructions. The quality and quantity of isolated RNA were measured using 2100 Bioanalyser  (Agilent)  and RNA 6000 Pico Kit reagents (Agilent). RNA samples with RNA integrity number (RIN)>7 were used for library construction using the 3′ mRNA-Seq Library Prep Kit FWD for Illumina (QuantSeq-LEXOGEN) as per the manufacturer's instructions. Amplification was controlled to obtain optimal unbiased libraries across samples by assessing the number of cycles (ranging from 20-24) required by qPCR. DNA High Sensitivity Kit for bioanalyzer was used to assess the quantity and quality of libraries, according to the manufacturer's instructions (Agilent). Libraries were multiplexed and sequenced on an Illumina Nextseq 500 at the genomics facility of IMBB FORTH according to the manufacturer's instructions.