GSM8846417: SCDi, CD45 CELLS; Mus musculus; RNA-Seq
For scRNAseq, 3 mice per experimental group were pooled into 1 sample prior to FACS-sorting to account for inter-animal variability. Each brain pool was sorted into 2 cell populations: tumor cells (DAPI-/CD45-/GFP+) and immune cells (DAPI-/CD45+/GFP-). Within 30 minutes after sorting, 3' scRNA-seq libraries were generated using a 10X Genomics Chromium controller. Libraries were then sequenced on a NovaSeq 6000 platform using a NovaSeq S2 flow cell (Illumina). Data analysis was performed in R studio (v1.3.1073) using Seurat (v4.1.1). Library preparation was performed according to the manufacturer's instructions (Chromium™ Single Cell 3' v3 Protocol, 10X Genomics). Briefly, cells were resuspended in the master mix and loaded 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 in every single GEM was retrotranscripted to cDNA, which contains an illumina 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 appropriate-sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added, but the transcript read was only single-end.