GSM7527726: C2, cord, SCI, 7dpi; Mus musculus; RNA-Seq
Blood was collected via cardiac puncture. Mice were then perfused with HBSS containing 0.2% heparin and the T8-T10 spinal cord was dissected into eppendorf tubes containing 500μl HBSS. Spinal cord was mechanically homogenised, incubated with papain enzymatic cocktail (L-glutamine-containing Leibovit's L-15 Medium with 0.1% papain and 0.1% DNAse I) for 20 minutes at 37°C on a shaker insubator, and filtered through a 40μm filter. 350μl blood was mixed 1:3 with 4mM EDTA and then incubated with red blood cell lysis buffer for 7 minutes at room temperature. Blood and cord samples were then stained with an anti-human CD45 APC-Cy7 antibody for 20 minutes on ice (in FACS buffer containing 0.5% BSA in 2mM EDTA with 1μg/μl mouse IgG for Fc-receptor blocking). huCD45+ were isolated from the spinal cord using anti-Cy7 magnetic beads for MACS sorting. Propidium iodide was added as a dead cell marker. Live huCD45+ were FACS sorted into eppendorf tubes containing 500μl ice cold PBS + 10% FBS. Library was performed according to manufacturer's instructions (single cell 3' library and gel bead kit v3). Briefly, huCD45+ cells were resuspended in the mastermix and loaded together with partitioning oil and beads into the chip to generate the gel bead-in emulsion (GEM). The polyA RNA from every bead was reverse transcribed to cDNA. Denatured libraries of the pooled DNA were sequenced on an Illumina NextSeq-500. Read1 supplied the cell barcode and unique molecular identifier (UMI), i7 the sample index, and Read2 the 3' sequence of the transcript. All samples were processed in one sequencing run.