GSM6914113: CD45+ immune cells, WT, scRNAseq; Mus musculus; RNA-Seq
Mice were transcardially perfused with ice-cold HBSS and the white matter of the lumbosacral spinal cord was dissected and dissociated using the adult brain dissociation kit (ABDK, Miltenyi Biotech) combined with manual mechanic dissociation and the translation and transcription inhibitors actinomysin D, triptolide, and asinomycin. Cells were strained through a 70 um cell strainer and debris isolated using the debris removal solution in the ABDK kit. Then, the cells were labelled with CD45+ microbeads (Miltenyi Biotech) and isolated through a magnetic column. 16K CD45+ cells were loaded into the 10x chip to achieve 10K cell recovery. Library was performed according to the manufacter's instructions (single cell 3' v3.1 dual index protocol, 10x Genomics). Briefly, CD45+ cells were resuspended in the master mix and loaded together 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 single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.