title: GSM6761302: PFC_WT_M, 10X single-nuclei RNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Control mice (Vgat-cre positive) were live-decapitated at eight weeks of age. Brains were rapidly extracted into a supplemented 4˚C Hibernate A medium, with GlutaMAX supplement (Fisher), B27 supplement (Fisher), and NxGen RNase inhibitor (0.2U/uL, Lucigen), and incubated for 30 seconds. The brain was sectioned into 1 mm sections using a brain matrix. The prefrontal cortex (PFC) was dissected with fine forceps, flash frozen on dry ice, and stored at -80oC. The nuclear isolation protocol was modified from Savell et al., 2020. On the day of nuclear dissociation, the frozen tissue was slowly thawed on ice and chopped with a scalpel blade 100 times in two orthogonal directions on a glass petri dish. The chopped tissue of three PFCs were pooled and transferred to a hypotonic 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, and the tissue was triturated ~30 times/sample using three glass pipettes of decreasing diameter. The tissue was then 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/uL 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 Aria II. Samples were counted and diluted to 1500 nuclei/uL before immediate 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 Sequencing Core at MUSC.