GSM8758542: UT_22q_M_1; Mus musculus; RNA-Seq
Mice were anesthetized by CO2 and transcardially perfused with 1X PBS. Brains were either frozen for whole brain sectioning/western blotting/qPCR or kept in RPMI (1X) + GlutaMAX (Gibco) + 1X MEM NAA for isolation of BBB-ECs. Data was initially analyzed separately and upon confirmation that there was no effect of sex, data from males and females were combined. Brains ECs were isolated using a protocol adapted from Munji et al., 2019 for sequencing experiments. For RNA sequencing, 3 brains of the same sex, genotype and treatment conditions were pooled into each sample (n = 3 samples per treatment condition). Brains were minced in PBS and subsequently digested in papain (Worthington) at 35o C for 90 minutes. Brains were triturated in trypsin inhibitor (ovomucoid; Worthington) and the cell suspension was digested in collagenase (Worthington) and neutral protease (Worthington) for 30 minutes at 35o C . Myelin was removed through density centrifugation with 22% Percoll. Cells were stained with FITC antimouse CD45 (Biolegend, 1:50), Zombie Violet (Biolegend 1:500), PE anti-mouse PDGFRb (Biolegend, 1:100), and APC-Cy7 anti-mouse CD31 (Biolegend, 1:50) for 30 minutes at 4o C in PBS. Live endothelial cells (CD31+ , CD45- , PDGFRb- ) were isolated by sorting on an Influx Cell Sorter (BD Biosciences). Cells were sorted into RNA lysis buffer (RLT buffer, Qiagen) and total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen) and sample quality was assessed on 4200 TapeStation (Agilent) as previously performed. mRNA library was prepared using the SMART-Seq HT kit (Clontech). Illumina indexes were added to cDNA using the Nextera XT DNA library preparation kit (Illumina). RNA-seq libraries were subjected to single end 75 bp read sequencing on an Illumina NextSeq 500 sequencer by Novogene (Beijing, China). Analysis was performed as previously described. In brief, processed reads were de-duplicated and mapped to the mouse transcriptome using Salmon with default settings and using the Ensemble mouse GRCm38 cDNA annotation as a reference transcriptome.