Amplicon sequencing of mus musculus: non-targeted adult mouse cortical brain microvessels
Genomic DNA was extracted from the isolated cortical microvessels using the DNeasy Blood and Tissue Kit (Lee et al. Nature Protocols 2019). Amplicons corresponding to the region of interest were then amplified by PCR using the Phusion High Fidelity Polymerase (New England Biolabs). The PCR amplicons were used for DNA library preparation. DNA Library Preparation, clustering, and sequencing reagents were used throughout the process using NEBNext Ultra DNA Library Prep kit following the manufacturers recommendations (Illumina, San Diego, CA,USA). End repaired adapters were ligated after adenylation of the 3 ends followed by enrichment by limited cycle PCR. DNA libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA,USA), and were quantified using Qubit 2.0 Fluorometer(Invitrogen, Carlsbad, CA)and multiplexed in equal molar mass. The pooled DNA libraries were loaded on the Illumina instrument according to manufacturers instructions.The samples were sequenced using a 2x250 paired-end (PE) configuration.