title: GSM7246175: cHET_F; Mus musculus; RNA-Seq
library_construction_protocol: 8-week old Mef2c cHetVgat and littermate control mice (Vgat-cre positive) were live-decapitated, and brains were rapidly extracted into ice-cold 4˚C Hibernate A medium, with GlutaMAX (Fisher), B27 (Fisher), and NxGen RNase inhibitor (0.2U/µL, Lucigen), and incubated for approximately 30 seconds. The brain was sectioned into 1 mm sections using a brain matrix and cold razor blades. The PFC was isolated with fine forceps and collected, flash frozen on dry ice, and stored at -80oC until the day of nuclear isolation. Bilateral prefrontal cortices were collected separately from three mice per group. We modified the nuclear isolation protocol described in Savell et al., 2020. On the day of nuclear isolation, the frozen tissue was thawed on ice and chopped with a scalpel blade 200 times in two orthogonal directions on a glass petri dish atop ice. The chopped tissue were then pooled per group and transferred into a lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL (Sigma) in Nuclease-Free Water). Supplemented Hibernate A medium was added to the lysis buffer to quench the reaction, and the tissue was triturated ~30 times/sample using three glass pipettes of decreasing diameter. This trituration cycle was repeated two more times. The mixture was filtered using a 40 micron filter (Fisher). The nuclei were isolated using 500xg centrifugation and washed with 1x PBS+1.0% BSA and 0.2U/µL RNase inhibitor. Nuclei were incubated with 7-aminoactinomycin D (7-AAD) (Invitrogen) for five minutes and sorted for 7-AAD-positive single nuclei using fluorescence-activated nuclear sorting on the BD FACS Aria II. Samples were counted and diluted to 1500 nuclei/µL before processing on the 10x Genomics Single-Cell Protocol by the MUSC Translation Science Lab. Libraries were constructed using the Chromium Single Cell 3' Library Construction Kit (10x Genomics, v3.1) and sequenced at Vanderbilt's Next Gen Sequencing Core (Illumina NovaSeq 6000). Droplet-based snRNA-seq libraries were prepared using the Chromium Single Cell 3' v3.1 according to the manufacturer's protocol. Libraries and sequencing were performed at the Translational Science Lab at MUSC.