GSM8117718
Fresh frozen brain tissue (10 sections of 1cm2 with a thickness of 40µm) were cut and collected and lysed in a sucrose lysis buffer (10 mM Tris-HCL (pH 8.0); 320 mM sucrose; 5 mM CaCl2; 3 μM Mg(Ac)2; 0.1 mM EDTA; 1 mM dithiothreitol (DTT) and 0.1% Triton X-100). Lysates were then filtered through a 70 µm cell strainer. Nuclei were purified by ultracentrifugation (107,000× g for 1.5 h at 4 °C) through a dense sucrose buffer (10 mM Tris–HCL (pH 8.0); 1.8 M sucrose; 3 μM Mg(Ac)2; 0.1 mM EDTA and 1 mM DTT). The supernatants were removed and pellets were re-suspended in 2% BSA/PBS containing RNase inhibitor (0.35 U/μL). The nuclei were incubated with fluorescently-conjugated antibodies directed against the neuronal marker NEUN (RBFOX3/NEUN (1B7) AF647 mouse mAB, Novus Biologicals, NBP1-92693AF647) and the transcription factor OLIG2 for the oligodendrocyte lineage (Anti-OLIG2 clone 211F1.1 AF488 mouse mAb, Merck Millipore, MABN50A4). After washing, the DNA dye DAPI was added and nuclei were sorted on a MoFlo Astrios. For each sample, we collected DAPIposNEUNnegOLIG2neg nuclei for snRNAseq Library construction was performed to the manufacturer's instuctions (single cell 3' v3.1 single index, 10X Genomics). Briefly, nuclei were resuspended in the master mix and loaded together with partioning oil and gel beads to form gel bead-in-emulsion (GEM). tThe poly-A RNA from the nucleus lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, unique molecular identifier (UMI) na dht e 10X barcode. The pooled barcoded cDNA was then cleaned with Silane Dynabeads, amplified by PCR and apropriated size fragments were selected using AMPure XP beads for subsequent library contruction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.