GSM9021010: Niacin Treated Mouse 1; Mus musculus; RNA-Seq
Mice were euthanized four weeks post-tumor implantation, and brains were isolated. To enrich for tumor tissue, brains were hemisected, and a 2 mm section around the tumor injection site was collected into Hank's Balanced Salt Solution (HBSS, Gibco). Four brain tissues were pooled together per sample to ensure an adequate number of cells for downstream scRNA-seq analysis (Control: 5 mice, one replicates; niacin injection: 10 mice, two replicates). Subsequently, brain tissues were homogenized using a Dounce homogenizer and filtered through a 40-µm cell strainer. The cell suspension underwent Percol gradient separation to remove myelin debris. After further filtration through a 35-µm cell strainer, cells were resuspended in PBS containing 1% heat-inactivated FBS (Sigma) and 0.1LJmM EDTA. Once a single cell suspension was generated as described above, cells were labelled with SYTOX™ Red Dead Cell Stain (ThermoFisher, S34860) to discriminate between live/dead cells. Live cells were sorted on FACSAria III (BD) using a 100-µm nozzle. scRNA gene expression libraries were prepared using the Chromium Next GEM single cell 3' reagent kits v3.1 from 10x Genomics. Appropriate volume of cells, as determined from the user guide for recovery of 3000 cells, was loaded on the Chromium single cell controller chip. Post cDNA amplification QC and quantification as well as library construction QC was done using an Agilent Bioanalyzer high sensitivity DNA chip for use with the Agilent 2100 Bioanalyzer. For sequencing, all libraries were pooled and loaded at 300pM on the NovaSeq Ilumina system using the NovaSeq S1 flowcell. A 28 base-pair Read 1 was used to sequence the cell barcode and UMI, an 8 bp i7 index read was used to sequence the sample index and a 91bp Read 2 was used to sequence the transcript using paired-end sequencing.