Whole cortex and brain endothelial cell-specific RNA-seq of mice subjected to peripherally-induced inflammation
Whole cortex and brain endothelial cell-specific RNA-seq of mice subjected to peripherally-induced inflammation
Mice were culled by cervical dislocation and the brain was quickly removed and cut into 1.5 mm coronal slices using a rodent brain matrix. To obtain enough RNA, each biological sample contained PFC from two mice. Brain slices were placed in cold RNase free PBS and PFC was further dissected and collected for RNA extraction. The dissected tissue was homogenized using a dounce tissue grinder (Kimble Cat# 885303-002) in 1 mL of homogenization buffer (47 mM Tris, 94 mM KCl, 11.3 mM MgCl2, 1%NP-40, 1mM DTT, protease inhibitor (Sigma Cat# P8340), 200 U/mL of RNasin (Promega Cat# N2115), 100 μg/mL cycloheximide, 1 mg/mL heparin). The homogenate was centrifuged (10,000 g / 10' / 4°C) to obtain a clear lysate, 10% (100 μL) of which was used to extract whole tissue RNA (Input). The remaining lysate (IP) was incubated with mouse anti HA-antibody (1:200, Biolegend Cat# 901514) for 4 hrs at 4°C in rotation, followed by addition of 200 μL of Pierce protein A/G magnetic beads (ThermoFisher Cat#88803) and incubation overnight at 4°C in rotation. The next day, beads were washed three times using high salt solution (50 mM Tris, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT and 100 μg/mL).  RNA was purified using RNeasy Plus Micro kit (Qiagen Cat# 74004). The concentration and quality of the RNA was assessed with an Agilent 2100 Bioanalyzer and only RNA samples with a concentration higher than 4 μg/μL and a RNA integrity number (RIN) greater than 7 were further sequenced.  Sequencing libraries were prepared using TruSeq RNA stranded mRNA kit (Illumina Cat# 20020594).