title: GSM4432649: EC6; Homo sapiens; RNA-Seq
library_construction_protocol: Isolation of nuclei was performed similarly as described in Mo et al. [PMID: 26087164]. Briefly, frozen brain tissue was dounce homogenized in 5 ml of lysis buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, pH 7.8, 1 mM DTT, 0.15mM spermine, 0.5 mM spermidine, 1X protease inhibitor (Sigma, 4693159001), and RNAse Inhibitor (Promega, N2615)). Following initial dounce homogenization, IGEPAL-630 was added to a final concentration of 0.3% and the sample was homogenized with 5 more strokes. The solution was then filtered through a 40 um cell filter and mixed with Optiprep (Sigma, D1556-250ML) to create a 25% Optiprep solution. This solution was then layered onto a 30%/40% Optiprep gradient and centrifuged at 10,000g for 18 minutes using the SW41-Ti rotor. The nuclei were collected at the 30%/40% Optiprep interface. Droplet-based single-nuclues RNA-sequencing (snRNA-seq) was performed using the Chromium Single Cell 3′ Reagent Kits v2 from 10X Genomics. Nuclei were resuspended to a concentration of 1000 nuclei/uL in 30% Optiprep solution before loading according to manufacturer's protocol, with 10,000 nuclei recovered per sample as the target. cDNA fragment analysis was performed using the Agilent 4200 TapeStation System. Sequencing parameters and quality control were performed as described by The Tabula Muris Consortium [PMID 30283141]. 
attributes.tag: GEO Accession
attributes.value: GSM4432649