GSM8389976: Cell multiplexed transcripts from 4 males rat at 4 weeks of age 2 (CTRL and 2 OI); Rattus norvegicus; RNA-Seq
Hindlimb muscles were removed from 4 weeks old CTRL and OI rats and dissociated using the gentleMACS dissociator (Miltenyi Biotech). After the digestion the mononuclear cells were filtered through a 100 μm nylon strainer, washed with FACS buffer (2% FBS in PBS), and the red blood cell were removed (Biolegend, Lysis buffer, 420301). Cells were labelled with Cell Multiplexing Oligos (CMOs 305, 306, 307 and 308), pooled and negative lineage selection was performed by FACS sorting using CD45 (Biolegend, 202205), CD31 (Abcam, ab33858), and CD11B (Biolegend, 201805) FITC conjugated antibodies . Prior to the FACS, cells were washed and passed through 30 µm filters. After the FACS cells were manually count and viability assess through trypan blue coloration to ensure a good viability (75-80%). Libraries were prepared using the 3' CellPlex Reagent Kits v3.1 (10x Genomics; Pleasanton, CA, USA) according to the manufacturer's instructions. Generated libraries were sequenced on an Novaseq6000. Briefly the cells were resuspended in the master mix and loaded with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every GEM was retrotranscripted to cDNA which contains an illumina primer sequence, unique molecular identifier (UMI) and the 10x barcode. The pooled barcoded cDNA was then cleanup with DynaBeads, amplified by PCR and the appropriate fragments size were seleted with SPRIselect reagant for subsquent library construction. During the library construction adaptor ligation mix and sample index were added.