title: GSM8881752: cortical microglia from adult, WT; Mus musculus; RNA-Seq
library_construction_protocol: 8-week-old mice were sacrificed by cervical dislocation, cerebral cortex from 5-6 mice (for each genotype) were isolated and immersed in 3 mL cooled HBSS, and then digested in 10 mL L15 culture medium supplemented with 80 k unit DNase I, 165 U papain  and 2 mg Cysteine at room temperature for 30 min. L15 culture medium supplemented with 10% fetal bovine serum was used to terminate the ingestion. After filtration through 70 μm cell strainer, cell mixture was centrifugated at 300 ×g at 18°C for 5 min. Cell pellets were resuspended by 4mL 30% Stock Isotonic Percoll (SIP) diluted in HBSS and followed by centrifugation at 300 ×g at 18°C for 40 min. Cell pellets were resuspended by 1 mL 2% FBS in PBS and incubated with Brilliant Violet 421™ anti-mouse CD45 Antibody and FITC anti-mouse CD11b antibodies for 30 min. Followed by rinsed for twice and fluorescence-activated flow cytometry (BD FACSARIASORP), CD45 and CD11b positive cells were obtained in 5% FBS in PBS. Library was performed according to the manufacturer's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GemCodes were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the Gel bead-in-EMulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. Sequenced samples were then processed using the Cell Ranger v6.0 and aligned to the GRCm39 as in RNA-seq. Seurat was used to quality control, filter unwanted cells (gene count<500 per cell; UMI counts > Infinity per cell; the percentage of mitochondrial genes > 25%). Followed by gene expression normalization for each cell via LogNormalize, cells were clustered based on principal component analysis and visualized by Uniform Manifold Approximation and Projection (UMAP). To identify DE genes for a single cluster, likelihood-ratio test was performed with following criteria: p value ≤ 0.01; log2fold change ≥ 0.26; the percentage of cells where the gene is detected in specific cluster > 10%. Top 10 DE genes were identified as the marker genes in specific cluster.