RRBS of mus musculus: old control microvessels
Genomic DNA was isolated from brain microvessels using the DNeasy Blood & Tissue Kit (Qiagen). DNA concentration was measured with the Qubit dsDNA BR Assay Kit (Thermo Fisher), and DNA quality was assessed by the Fragment Analyzer and the DNF-488 High Sensitivity genomic DNA analysis kit (Agilent). DNA Methylation Profiling (RRBS Service) was performed by Diagenode. Each experimental group for RRBS contained three biological replicates. RRBS libraries were prepared using the Premium Reduced Representation Bisulfite Sequencing (RRBS) Kit, with 100 ng of genomic DNA per sample used to start library preparation. PCR clean-up after the final library amplification was performed using a 1x (1.45 for second run) beads:sample ratio of Agencourt AMPure XP (Beckman Coulter). The quality of the RRBS library pools was evaluated by measuring their DNA concentration with the Qubit dsDNA HS Assay Kit (Thermo Fisher), and the profile of the pools was verified using the High Sensitivity DNA chip for 2100 Bioanalyzer (Agilent) or DNF-474 NGS fragment kit on a Fragment Analyzer (Agilent). Deep sequencing of RRBS library pools was performed with a NovaSeq6000 (Illumina) using 50 bp paired-end read sequencing (PE50).