Smartseq3 Single-cell RNA-seq analysis of acutely FACS-purified tdTomato+ astrocyte derived cells from aged injured mice in vivo

To investigate the mechanisms that underlie astrocyte dedifferentiation, we performed single cell RNA sequencing analysis of tdTomato+ astrocyte derived cells approx 1 year after stab-wound injury. 2-3 month old GFAP-CreERT2;p53flox/flox;LSL-tdTomato (p53Gfap-icKO) mice were subjected to intracortical endoxifen injection to enable deletion of p53 gene and simultaneous expression of tdTomato for fate-mapping specifically in astrocytes (GFAP+) in the context of a stab-wound injury. GFAP-CreERT2;p53wt/wt;LSL-tdTomato (p53Gfapwt/wt) mice were used as control whereby p53 remains fate-mapped tdTomato+ astrocytes remain p53 wildtype after endoxifen injection. Approx 10-12 months after injury, mice were sacrificed and the peri-wound region was dissected. Mice of the same genotypes were pooled (3 p53Gfap-icKO and 2 p53Gfapwt/wt) and digested using the Miltenyi Adult brain dissociation kit. Single cell suspension was FACS sorted for tdTomato+ cells into 384 well plates and then Smartseq3 libraries were generated. Overall design: FACS-purified tdTomato+ cells from 3 pooled p53Gfap-icKO brains or 2 pooled p53Gfap-wt/wt brains with raw data for 145 KO and 125 WT.