title: GSM8653655: Experiment2_Gene_expression1: scRNA-seq from the V-SVZ; Mus musculus; RNA-Seq
library_name: GSM8653655
library_construction_protocol: Samples from mice injected with Ad:Cre:GFP were prepared as following:  15-24 hours after Ad:Cre:GFP intraventricular injections, mice (Experiment 1: n=14, P27-30, Experiment 2: n=12, P27-p30) received intraperitoneal administration of 2.5% Avertin followed by decapitation. Brains were extracted and 0.5 mm slices were obtained with a mouse brain slicer (Steel Brain Matrix - Coronal 0.5mm, Alto). The V-SVZ lateral wall and dorsal wedge were microdissected and considered separate samples. To analyze the transcriptomic profile of aged mice we also microdissected the V-SVZ of two non-infected hGFAP:GFP 1 year old mice. The wedge region was not included. All V-SVZ micro-dissections were performed in L-15 medium on ice and the tissue was transferred to Papain-EBSS (LK003150, Worthington). Tissue was digested for 30 min at 37ºC in a thermomixer at 900 RPM. Mechanical dissociation with a P1000 pipette tip (20 seconds), then with a  fire-polished pasteur pipette for 3 min.  Cells were centrifuged for 5 min, 300 RCF at room temp, and the pellet was resuspended with DNAase/ovomucoid inhibitor according to the manufacturer's protocol (Worthington). Cells were incubated in red blood cell lysis buffer (420301, Biolegend) 3-4 min at 4ºC. For MULTI-seq barcoding, cells were suspended with Anchor:Barcode solution for 5 minutes at 4ºC. A Co-Anchor solution was added and incubated for 5 min87. Samples were combined and filtered with a FlowMi 40µm filter (BAH136800040-50EA, Sigma). To remove myelin, the cell suspension was incubated with Myelin Removal Beads (130-096-733, Miltenyi Biotec) (6μl/brain) for 15 min at 2-8°C. Cells were washed with 0.5% BSA-PBS and transferred to MACS columns. The effluent was collected, and the cell density was counted. Cells were loaded into a 10x Genomics Chromium Single Cell Controller. We used the 10x Genomics Chromium Single Cell 3' Library & Gel Bead Kit v3.1 to generate cDNA libraries for sequencing according to the manufacturer's protocols. MULTI-Seq barcode libraries were prepared according to MULTI-Seq (McGinnis et al. 2019)  protocols.