GSM8058322: wt hippocampus; Mus musculus; RNA-Seq
Prior to hippocampal collection, animals (57 weeks old GlaKO and littermate controls) were anesthetized using a mix of xylazine 10 mg/kg and ketamine 100 mg/kg and transcardially perfused with PBS to washout the blood. Hippocampi were dissected in PBS on ice and dissociated using the Adult Dissociation Kit (130-107-677, Miltenyi Biotec). Briefly, hippocampi were transferred into a gentleMACS C tube containing 1900 μl of buffer Z and 50 μl of enzyme P. Before attaching the C tube to the gentleMACS Octo Dissociator, 20 μl of buffer Y and 10 μl of enzyme Z were added. The suspension was passed through a MACS SmartStrainer (70 μm) placed on a 50 mL tube on ice. Cells were centrifuged at 300xg for 10 min at 4°C and resuspended in 1550 μl of cold PBS. To remove cell debris after dissociation, 450 μL of Debri Removal Solution were added to prepare a density gradient, overlaid with 2 mL of cold PBS. After centrifugation at 3000xg and 4°C for 10 min, the two top layers were discarded and the pellet was washed and resuspended in Dounce buffer [15 mM HEPES, 0.6% (w/v) Glucose in Hank's Balance Salt Solution, H8264, Sigma). Viable cells exclusion was performed by FACS cell sorting (FACSAria, BD Biosciences) using 7-AAD dye (1 μg/mL, V35123, Invitrogen). Cell suspensions were processed to generate stable cDNA libraries. For every sample, 10'000 cells were loaded into a Chromium Single Cell A Chip (10x Genomics) and processed according to the manufacturer's instructions. The sequencing library was generated with the 10x Genomics Chromium and Single Cell 3` Library v3.1 technology was used for single cell barcoding. Sequencing of the library pool was performed on the Illumina NovaSeq 6000 next generation sequencing system with asymmetric paired-end reads. Demultiplexing of the Illumina sequencer's base call files (BCLs) into FASTQ files was performed using the 10x Genomics package Cell Ranger V.4.0 with the mkfastq pipeline.