GSM9323665: 6_S04269_TD_replicate2; Homo sapiens; RNA-Seq
Dissected prefrontal cortex was preserved at -80C until processing. The specimens were gently thawed in 2 ml of ice-cold phosphate-buffered saline (PBS, devoid of Ca2+ and Mg2+). This solution was supplemented with various compounds to preserve the quality of the sample: 0.54 μM Nectostatin (Inc. #J65341, ThermoFisher Scientific Chemicals, Waltham, MA), 1 μM HPN-07 (SML2163, Sigma-Aldrich), 0.32 μM sodium hydroxybutyrate (A1161314, ThermoFisher Scientific Chemicals), 78 nM Q-VD-Oph (SML0063, Sigma-Aldrich), and 0.2 U/ml of Ribolock RNase inhibitor (EO0381, Life Technologies Corporation, Carlsbad, CA). The tissue, held over dry ice in a 60-mm Petri dish, was methodically minced into fragments smaller than 2 mm using a razor blade. Subsequently, this tissue was transitioned to a 2 ml Dounce homogenizer containing 1.5 ml of nuclei extraction buffer (130-128-024, Miltenyi Biotec, Bergisch Gladbach, Germany) along with 0.2 U/ml Ribolock RNase inhibitor.  Samples were then centrifuged at 600×g for 5 min, resuspended in 400 μL of PBS enriched with 2% BSA, passed through a 70-μm FLOWMI for clarification, and strained via a 40-μm FLOWMI. Specimens were then entrusted to the Hunstman Cancer Institute High Throughput Genomics Core for 10X Genomics library construction and sequencing. Libraries were prepped according to the 10X Genomics scRNA-seq 3' v3.1 Next GEM protocol, NovaSeq Reagent Kit Series 10B_150x150 bp Sequencing.