GSM8449847: 42dpi, scRNAseq; Mus musculus; RNA-Seq
To isolate astrocytes, brains were intracardially perfused with sterile 1x PBS at respective time points and placed in RPMI media (Gibco, 11875101) containing 1% Fetal Bovine Serum (FBS) (Corning, MT35015CV) and 25 mM HEPES. Perfused brains were transferred to a chilled 60 mm petri dish, dissociated with a razor blade, and treated with 4 ml of 0.25% trypsin. Brain suspension was digested at 37C for 30 minutes in 50 ml conical tubes. During digestion, the mixture was inverted every 5 minutes. Trypsin digestion was stopped with the addition of 30 ml of RPMI containing 20% FBS and centrifuged at 1000 RCF (g) for 5 minutes at 4C. Supernatant was decanted, and the pellet resuspended in 1% FBS RPMI to a total volume of 7 ml. The suspension was transferred to a 15 ml conical tube with 3 ml of 100% Percoll (Sigma Aldrich, P1644), gently mixed and underlaid with 1 ml of 70% Percoll made in 1% FBS RPMI. Samples were centrifuged at 2500 RCF (g) for 20 minutes at 4C. Following density separation, glial cells were harvested from the interphase and washed in 1% FBS RPMI and resuspended in FACS buffer for flow cytometry staining. To identify transcriptional astrocyte subsets, single cell RNA sequencing (scRNAseq) was performed from murine cells. To obtain a pure astrocyte sample, extracellular staining with anti-astrocyte cell surface antigen-2 (ACSA-2) was performed (Miltenyi Biotec, 130-117-535). The viability stain 7-aminoactinomycin (ThermoFischer, A1310), was added to the FACS buffer to identify dead cells. ACSA-2+ astrocytes were sorted and collected in a flow tube containing 10% FBS complete astrocyte RPMI with the Beckman Coulter MoFlo Astrios EQ Cell Sorter. For reporter experiments, sorting was performed on live cells via DAPI and tdTomato expression to collect live Lcn2CreERT2+ RAs for sequencing. A maximum of 16,000 astrocytes from each time point following the astrocyte cell suspension protocol was collected and pre-loaded on a 10X Chromium chip and Chromium controller for barcoding. Single cell RNA sequencing steps from the 10X Genomics 3' kit (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 PN-1000128) were followed according to supplier instructions to extract RNA for sequencing Cell purity throughout preparation was verified on the Agilent 2100 Bioanalyzer following cDNA amplification and library construction. When submitting samples to the 10X Genomics Core at UCSD, 1 billion reads per sample were requested to achieve a saturation level above 90%. After sequencing, the Cell Ranger program (v5.0.1) was utilized to align reads from the sequencing fastq data files to a mouse reference genome mm10 and generate gene-barcode expression matrices. Mean reads and median genes per cell were reviewed for statistical relevance and then aligned sequences were uploaded to R (v4.3.2) package to generate plots and obtain gene statistics per cell and cluster.