title: GSM8232404: Brain, Melanoma+Isotype; Mus musculus; RNA-Seq
library_name: GSM8232404
library_construction_protocol: After removal of CNS, tissues was flash frozen in liquid nitrogen and stroed at -80 C. For isolation of nuclei, briefly, CNS tissue was homogenized in Nuclei EZ lysis buffer (Millipore Sigma) and upon incubation was filtere through 70µm filter. The suspension was centrifuged at 600g for 5 min at 4°C. The pellet was washed with nuclei wash and resuspension buffer (1x PBS, 1.0% BSA, 0.2 U/μl RNase Inhibitor) and centrifuged at 600g for5min at 4°C.Following this, Nuclei were stained for DAPI, NeuN and Olig 2 markers and sorted by Flow cytometry. Nuclei were pre-gated on DAPI signal. NeuN and Olig2 negative nuclei were collcted and used as input for snRNA-seq. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs 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.