GSM7065625: Sca1_AAV_NC; Mus musculus; RNA-Seq
Veingrafts were harvested and digested. After full digestion, cell pellets were suspended in PBS and stained with a LIVE/DEAD ™ Fixable Near-IR Dead Cell Stain Kit (1:1000) and Hoechst 33342 (Invitrogen, H3570, 1:1000). After washing using PBS, cells were resuspended in PBS, after which single nucleated live cells (Hoechst+ & Dead Cell Stain-) were sorted into PBS with 0.04% BSA using a BD FACS ARIA II Flow Cytometer (BD Biosciences). For CD45- cells, a CD45 antibody (Invitrogen, 14-0452-82, 1:2000) was additionally used. For RFP+ cells, FACS sorting was based on their autofluorescence. Samples were subjected to scRNA-seq. A ChromiumTM Single Cell 3' Reagent Kit v2 or v3 chemistry (10x Genomics) was used and a standard protocol followed. The library was generated and sequenced on a Novaseq6000 PE150 platform (Illumina) with paired-end 150 bp sequencing strategy.