GSM8443496: Run 4: combined group with 2d, 4d, 7d and naïve astrocytes; Mus musculus; RNA-Seq
At the appropriate timepoint post-FUS-BBBO (2, 4 or 7 days) animals were euthanized with naïve control animals by cardiac perfusion with sterile saline.  The hippocampi of each animal were dissected out and timepoint-matched male and female tissue was pooled.  The tissue was minced with a sterile razor and underwent enzymatic digestion according to the manufacturer's protocol using a Postnatal Neuron Neural Tissue Dissociation Kit (Miltenyi, # 130-094-802).  Following enzymatic digestion the tissue was filtered using a 70-μm cell strainer and pelleted by centrifuging at 300xG for 10 minutes at room temperature.  The pellet was then resuspended in 900 μL MACS buffer (Miltenyi, #130-091-376), and incubated with 100 μL Myelin II removal beads (Miltenyi, #130-096-433) for 15 minutes at 4°C, flicking to mix the solution every 5 minutes.  The cells were pelleted at 4°C by centrifuging for 1 minute at 9,300 RCF.  The supernatant was discarded, and the pellet was resuspended in 1 mL of MACS buffer.  This cell suspension was then passed through LS columns (Miltenyi, #130-042-401) on a magnet to extract the magnetic myelin beads.  The cells were pelleted again for 1 minute at 9,300 RCF, and resuspended in staining buffer, consisting of a 1:100 dilution of ACSA-II-PE conjugated antibody (Miltenyi, #130-123-284) and a 1:500 dilution of a unique hash tag antibody (Biolegend, #155801-8) for each timepoint group in 1xPBS.  Cells were incubated with this staining solution for 30 minutes at 4°C.  The cells were then pelleted, the supernatant was discarded, and the cells were resuspended in flow cytometry buffer consisting of a 1:1000 dilution of DAPI, 1:1000 Vybrant DyeCycle Ruby (Invitrogen, #V10309) and 1:500 DNase (from 4 mg/mL stock) in 1x PBS.  The cells were then taken to the Columbia Stem Cell Initiative Flow Cytometry Core and live, ACSA-II+ astrocytes were sorted for each condition.  These sorted cells were then brought to the Columbia Genome Center for single-cell RNA sequencing on a 10x Genomics Chromium Single Cell 3' flow cell. The 10x Genomics Chromium platform was used to generate low-depth data, which was procseed using the Cell Ranger analysis pipeline to align reads and generate feature-barcode matricies.