title: Profiling of the brain endothelial transcriptional changes upon Gpr126 depletion at P18
design: Profiling of the brain endothelial transcriptional changes upon Gpr126 depletion at P18
library_construction_protocol: WT and Gpr126iECKO ECs were derived from brains of pups at post-natal stage P18 upon tamoxifen treatment. Briefly, the brains were digested enzymatically in combination with gentle dissociation and adult brain dissociation kit [130-107-677]; Miltenyi Biotech). After dissociation, the myelin cell debris and erythrocytes were removed according to the manufacturer protocol. ECs were enriched by depletion of CD45-positive cells with CD45 microbeads (30-052-301; Miltenyi Biotech). After AN2 re-expression according to the manufacturer protocol, pericytes and astrocytes were depleted using AN2 microbeads (130-097-171; Miltenyi Biotech) and ACSA-2 microbeads (130-097-678; Miltenyi Biotech), respectively, followed by positive selection using CD31 microbeads (30-097-418; Miltenyi Biotech).  Total RNA was extracted from fBECs of WT and Gpr126iECKO mice at P18 using Maxwell RSC simplyRNA tissue kits with the Maxwell RSC Instruments (Promega Corporation). The amount of RNA was measured using the Nanodrop technique and its integrity assessed using Agilent Bioanalyzer 2100 with Nano RNA kits (RIN > 8). Libraries for RNA sequencing were prepared following the manufacturer protocols. mRNA-seq indexed library preparation was performed starting from 500 ng of total RNA with the Illumina ligation stranded mRNA kit according to the manufacturer instructions. Indexed libraries were quality controlled on an Agilent Bioanalyzer 2100 with High Sensitivity DNA kits, quantified with Qubit HS DNA, normalized and pooled to perform multiplexed sequencing runs.