GSM5016842: MG_WT_2; Mus musculus; RNA-Seq
The meninges were removed from the mice brains of different genotypes and dissociated using the Neural Tissue dissociation kit, papain based (Miltenyi Biotech) and GentleMACSdissociator (NTDK Brain setting). The lysate was collected and passed through a 70μm strainer to obtain a single cell suspension. Next, myelin was removed fromthe cells by incubating with magnetic myelin beads (Myelin Removal Beads II, human, mouse, rat, Miltenyi Biotech) and removed using the AutoMACs. Cells were then incubated with CD11b+ magnetic beads CD11b MicroBeads, human and mouse, (Miltenyi Biotech) and sorted using the AutoMACs. After sorting, cells were counted and diluted according to the specifications in the Single Cell 3′ v3.1 Reagent Kits User Guide (10x Genomics, Pleasanton, California) to capture about 4000 cells per sample. Single-cell RNA-Seq libraries were prepared per the Single Cell 3′ v3.1 Reagent Kits User Guide (10x Genomics, Pleasanton, California). Cellular suspensions were loaded on a Chromium Controller instrument (10x Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs). GEM-RT was performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA). GEMs were then harvested, and the cDNA was amplified and cleaned up with SPRIselect Reagent Kit (Beckman Coulter, Brea, CA).  Indexed sequencing libraries were constructed usingChromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified by quantitative PCR using the Collibri LibraryQuantification Kit (Thermo Fisher Scientific). Libraries were sequenced on a NovaSeq 6000 (Illumina, San Diego, CA) as per the Single Cell 3′ v3.1 Reagent Kits User Guide, with a sequencing depth of ~40,000 reads/cell. batching protocol: To minimize batch affect, samples were processed through the GEM-RT step in batches of three on three consecutive days. We attempted to mix the genotypes over all three days, however this was not done perfectly. In addition, the order each genotype was processed on a given day was also mixed, but again not perfectly. The process day and process order value is listed in the caracteristics column of the Samples Table. After all nine samples were taken to the GEM-RT stage, all samples were taken through the rest of the library construction protocol together on the same day. 10x Genomics Single Cell 3' v3.1