GSM7066314: Macrophge, B6_WT1; Mus musculus; RNA-Seq
WT C57BL/6, WT C57BL/10, mdx, B6A/J mice were humanely euthanized, perfused to clear circulating immune cells, and hind limb lymph nodes removed. Single-cell suspensions were prepared from healthy and dystrophic muscle by mechanical dissociation and enzymatic digestion with collagenase and DNase. Cell suspensions were washed and filtered sequentially through 70- and 40-micron cell strainers. Live muscle macrophages were FACS-sorted based on their expression of F4/80 and CD11b, and single-cell libraries constructed with the 10X Genomics platform. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs 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.