GSM8733694: 1167, Frontal cortex, mutant; Mus musculus; RNA-Seq
At P30, littermate Tsc2del/fl SynCre+ and Tsc2fl/fl SynCre- animals were euthanized with carbon dioxide. A piece of left frontal cortex was dissected and transferred to a dounce homogenizer with 1ml of homogenization buffer (HB) (250mM Sucrose (IBI Scientific IB37160), 25mM KCl (Sigma P9541-500G), 5mM MgCl2 (Sigma 63068-250G), 20mM Tricine-KOH (Tricine T0377-100G), spermine 150uM (Abcam ab146523), spermidine 500uM (Sigma S2501-1G), DTT 1mM (Sigma D0632-1G), pH 7.8), followed by 10 strokes of a “tight” pestle. Then 320ul of 5% IGEPAL in HB was added to the homogenate, followed by 5 more strokes with the pestle. The resulting homogenate was filtered through a 40um cell strainer and then washed with 4ml of buffer HB. This was mixed with 5ml of working solution (50% optiprep, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH, pH 7.8) to generate a nuclei suspension in 25% optiprep. A gradient was formed with (bottom to top) 1ml 40% optiprep (2ml working solution + 0.5ml HB + 4ul RNAsin (N2511; Promega)), 1ml 30% optiprep (1.5ml working solution + 1ml HB + 4ul RNAsin), and then 10ml of 25% optiprep with homogenate in ultracentrifuge tubes (16mm x 76mm UltraClear; Beckman Coulter). Gradients were centrifuged at 10,000g for 20 minutes. Nuclei were collected from the interface between the 30% and 40% optiprep layers. Nuclei suspension was washed once with PBS (Invitrogen 14190-250) + 1% (BSA EMD-Millipore 2930-100GM) + 5mM MgCl2 (Sigma 63068-250G). Single cell RNA-Seq was provided by the Single Cell Core at Harvard Medical School, Boston, MA. Library size was quantified on a Bioanalyzer (Agilent) and concentration was quantified by qPCR (Kapa Library Quantification – Complete kit ROX low; Roche Sequencing 07960336001).