GSM6910122: Dn1; Homo sapiens; RNA-Seq
For RNA sequencing, the peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation using Lymphosep, Lymphocyte Separation Media (#L0560-500, Biowest, France), density 1.077 g/ml. After that, CD14+ monocytes from PBMC fraction were obtained by FACS. Cells were resuspended in 150 μl of staining buffer (#2701005, Cell Staining Buffer, Sony, Japan). Monocytes were defined as CD45+CD56-CD14+7-AAD- population. Conjugated monoclonal antibodies to CD45, CD56, CD14, 7-AAD were added to the cell suspension. Samples were analyzed on a MoFlo XDP cell sorter (RRID:SCR_019665, Beckman Coulter, USA). Sorting of monocytes was carried out in the Purify 1-2 mode, the sorting efficiency was 70%, the purity of the target population was 96-99% RNA libraries were prepared with NEX 160 T flex Rapid Directional qRNA-SeqKit using indexed barcodes NEXTflex-qRNA-8nt-Barcodes (#NOVA-5198-02, Bioo Scientific, PerkinElmer Applied Genomics, USA) according manufacture`s protocols. Ribosomal RNA depletion was performed with NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E7400, New England Biolabs Inc., USA)