title: GSM4775579: NC16; Homo sapiens; RNA-Seq
library_construction_protocol: we placed frozen cortical tissues directly into a prechilled Dounce homogenizer with ice-cold homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM tricine-KOH [pH 7.8], 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, protease inhibitors, 5 μg/mL actinomycin, 0.32% NP40, and 0.04% BSA). After 25 strokes with a loose pestle, we mixed the homogenate 1:1 in OptiPrep and centrifuged the solution at 10,000 × g for 20 min at 4 °C. We subsequently collected the separated nuclei, which were pelleted at the bottom of the centrifuge tube, washed them once to remove the OptiPrep, and resuspended them in DMEM/F12 supplemented with 10% FBS. We diluted the nuclei to 40,000 nuclei per mL−1, ensuring this dilution by counting with a hemocytometer. We also assessed the purity of the single-nucleus suspensions by flow cytometry; this protocol routinely yielded high-purity, single-nucleus suspensions (i.e., >95% DAPI+ nuclei in all samples). All buffers and gradient solutions for nuclei extraction contained 60 U/mL RNAsin (Promega). We generated snRNA-seq libraries using the Chromium Single Cell 3′ Library Kit v3 (1000078; 10x Genomics) according to the manufacturer's instructions.