title: GSM7162894: 42358-7d; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanised with pentobarbitol, intercardiacally perfused with cold PBS, and brains were extracted. Tumor and/or treated lesion were disected from brains and minced in cold PBS for 5 minutes. For each sample, gentleMACS Octo Dissasociator tubes were prepared with 4.8 ml 2% FBS in PBS with 100 mg/ml collagenase/disapse (Sigma-Aldrich). Minced tissue samples were added to dissasociator tubes, then incubated for 30 minutes at 37ºC with constant low-speed rotation. Samples were then centrifuged at 300 g for 5 minutes at 4ºC and pellets were resuspended in 5 ml HBSS on ice. Cells were passed through a sterile 100 µm filter, centrigued again at 300 g for 5 minutes at 4ºC, and pellets were resuspended in myelin removal bead mixture (1800 µl FACS buffer with 200 µl myelin removal beads II (Miltenyi Biotec)), and incubated for 15 minutes at 4ºC. 18 mls of FACS buffer were added to each sample, centrigured at 300 g for 5 minutes at 4ºC, resuspended in 1 ml FACS buffer, and filtered through LS columns (Miltenyi Biotec). Samples were incubated with red blood cell lysis buffer for 15 minutes at room temperature, then centrifuged at 300 g for 5 minutes at 4ºC, and resuspended in 100 µm FACS buffer for 10X Chromium workflow. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, cells 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.