GSM5009113: LPS+Clu; Mus musculus; RNA-Seq
Brain endothelial cells were isolated by FACS sorting. 4-5 mice per group were anesthetized and perfused. The hippocampi from each group were pooled, minced and digested using the neural dissociation kit according to kit instructions (Miltenyi, 130–092-628). Myelin removal was performed using 0.9M sucrose in dPBS. Cell pellets were resuspended in FACs buffer and blocked for 5 min on ice with Fc preblock. followed by 30 minute staining with anti-CD31-PE/CF594 (1:75, BD 563616), anti-CD45-PE/Cy7 (1:200, BD 103114), and anti-Cd11b-FITC (1:100, BD 101206). Dead cells were excluded by staining with SYTOX™ Blue Dead Cell Stain (1:2000, Thermo S34857). Flow cytometry data and cell sorting were acquired on an ARIA 3.3 (BD Biosciences) with FACSDiva software (BD Biosciences). single cell suspensions were counted using the Countess automated cell counter (ThermoFisher). After counting, cells were loaded onto the 10x Chromium chip and using the recommended loading volumes and run on the 10x chromium v3 controller. Immediately after running the chip, the samples were reverse transcribed to cDNA according to the manufacturer's instructions and kept at 4ºC overnight. The following day, sample cleanup and cDNA amplification with 12 PCR cycles were performed according to instructions. The amplified cDNA was then checked for quality on the Agilent Bioanalyzer (HS DNA kit). Libraries were generated according to manufacturer's instructions using unique barcodes for each sample and the finalized libraries were again checked for quality on the Agilent Bioanalyzer (HS DNA kit). Samples were sent for sequencing to Novogene where they were first assessed for library quality on a single lane of the HiSeq System followed by deep sequencing on a Novaseq S4 flow cell to ensure > 20,000 reads/cell as recommended. scRNAseq (10x Genomics chromium v3)