biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsOwen DandoNextSeq 500RNA-seq of coding RNAMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14490The blood-brain barrier is an interface for signal exchange between the brain and the periphery. We reasoned that brain endothelial cells (BECs) are ideally positioned to receive signals from the blood and transduce them into the brain via direct communication with astrocyte endfeet. In combination with the a method to define the in-vivo protein composition of astrocyte endfeet, we used mouse brain endothelial cell-specific RNA-seq to study BEC-endfeet interactions in response to peripheral inflammatory insult by LPS.biostudies-arrayexpressNucleic Acid Extraction - 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.Sample Collection - 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).Library Construction - Sequencing libraries were prepared using TruSeq RNA stranded mRNA kit (Illumina Cat# 20020594).Sequencing - Libraries were sequenced on a NextSeq 500 system (Illumina).OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesOwen DandofalseWhole cortex and brain endothelial cell-specific RNA-seq of mice subjected to peripherally-induced inflammationThe blood-brain barrier is an interface for signal exchange between the brain and the periphery. We reasoned that brain endothelial cells (BECs) are ideally positioned to receive signals from the blood and transduce them into the brain via direct communication with astrocyte endfeet. In combination with the a method to define the in-vivo protein composition of astrocyte endfeet, we used mouse brain endothelial cell-specific RNA-seq to study BEC-endfeet interactions in response to peripheral inflammatory insult by LPS.2025-09-09T00:00:00Z2025-09-10T00:01:34.849Z2024-10-01T10:26:56.025ZE-MTAB-14490ERP164653EFO_0002944EFO_0004170EFO_0005518EFO_0003738EFO_0004184