The original VINE-seq protocol extracts nuclei from isolated microvessels by physically mashing microvessels through a cell strainer. While this approach yielded high-quality snRNA-seq data, snATAC-seq data was of poor quality, as assessed by TSS enrichment scores. We hypothesized that poor snATAC-seq data resulted from physical damage during the mashing process and optimized alternative chemical and enzymatic approaches to digest the microvessel basement membrane without harming the nuclei. After screening 12+ detergents, agents, and enzymes, we found success with Collagenase III (Cell Signaling Technology, 25970). After centrifugation of isolated microvessels, the supernatant was aspirated, and the remaining vessel pellet was resuspended in 1.5mL of digestion buffer (DB: 2mg Collagenase III in 1mL of sample buffer). The microvessels suspended in digestion buffer were transferred to a 2mL Eppendorf DNA Lo-Bind tube, and digested at 37C for 10 minutes with rotation at 150rpm/min. Following incubation, the vessel suspension was pipetted 20 times and filtered through a 40L strainer on a new 50mL conical tube pre-wetted with wash buffer 2 (WB2: 2% BSA in PBS with 1mM EDTA and 1:5000 RNase Inhibitor). Vessels were washed over the strainer with 40mL of WB2. Isolated vessel nuclei were centrifuged at 500g for 5 minutes with the brakes set to 2. Nuclei extracted from vessels as well as those originating from the parenchymal layer were transferred to FACS tubes, stained with 7-AAD (1:100, Sigma, SML1633) and rabbit monoclonal Anti-ERG antibody Alexa Fluor 647 (1:100, Abcam, ab196149), and nuclei collected on a BD FACSAria III into chilled tubes containing 1ml of NB without protease inhibitor. Each sample was sorted for vascular nuclei as well as ERG+ parenchymal nuclei, which captures microglia (PMC8784313), at a 3:1 ratio. This sorting strategy promotes adequate microglial representation. Sorted samples were inspected for lack of debris on a bright-field microscope. Nuclei were then prepared for transposition following the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + GEX assay kit protocol.For droplet-based snRNA and snATAC-seq libraries were generated using the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + GEX assay kit following the manufacturers protocol (CG000338), to prepare paired ATAC and GEX libraries from the same nuclei.