title: GSM6701351: Nav1 KO, snRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Brain tissue was obtained from aged-matched adult male Nav1 KO (n=4) and C57BL/6 (n=5) mice. The PFC was quickly dissected from the freshly extracted brain tissue and it was placed in chilled PBS with Hibernate AB Complete (HEB) medium (BrainBits LLC, Springfield, IL) at 4 0C. Freshly dissected PFC from each group were pooled in separate 50 ml conical tubes with a minimum amount of chilled Hibernate AB Complete (HEB) medium (BrainBits LLC, Springfield, IL). Then, 5 ml of chilled lysis buffer (2mM Tris-HCl, 2mM NaCl, 0.6 mM MgCl2 and 0.02% NonidetTM P40 Substitute in nuclease-free water) was added to the tubes, which were then incubated at 4 0C for 10 min. The amount of lysis buffer and the incubation time were optimized determining the amount of buffer and incubation time that produced high-quality nuclei. After adding 5 more ml of HEB medium to the tubes, the tissues were triturated with a fire-polished silanized Pasteur pipette for 10-15 passes and then strained with 30 um MACS strainer (Miltenyi Biotec Inc, Auburn CA). Nuclei were pelleted by centrifugation at 500xg centrifugation for 5 min at 4°C and were then resuspended in a chilled PBS with 1% BSA (Invitrogen AM2618, Thermo Fisher Scientific, Pittsburgh PA) wash buffer with 0.2 U/ul RNase inhibitor (#3335402001, Millipore Sigma, Darmstadt Germany). The nuclei were pelleted and resuspended twice, strained using a 30 um MACS strainer, and then centrifuged and resuspended in a chilled wash buffer. Mouse anti-NeuN antibody (MAB377, Millipore Sigma, Darmstadt Germany) was added at a 1:500 dilution, and the samples were incubated for 40 min at 4°C. Samples were then centrifuged at 400xg for 5 minutes at 4°C and the pelleted nuclei were resuspended with chilled wash buffer. Alexa 647 chicken anti-mouse antibody (A21463, Life technologies, Pittsburgh PA) was added to the preparations at a 1:500 dilution and the samples were incubated for 40 min at 4°C. After incubation, nuclei were pelleted and resuspended, and Hoechst 33342 (Invitrogen H3570, Thermo Fisher Scientific, Pittsburgh PA) was added to a final concentration of 0.25 ug/ml. Single nuclei sorting was conducted using a 6-laser BD Influx sorter (BD Biosciences, San Jose CA) with a 100-um nozzle in the Stanford Shared FACS Facility. Various control samples (unstained, Hochest33342+ or NeuN+) were examined to optimize the gating strategy. Nuclei were gated based on size, scatter properties and staining for Hoechst and NeuN. To ensure that we were able to analyze different types of cells, ~60% of the sorted nuclei were collected as NeuN+ and 40% were NeuN-. Single nuclei were sorted into collecting tubes and then visually inspected under a microscope for quality control; they were then pelleted and resuspended to produce a solution with 600 nuclei/ul in the final volume. Single nuclei were captured in droplets with barcodes using the 10x Genomics Chromium system and cDNA libraries were produced using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics, Pleasanton, CA) according to the manufacturer's instructions. The samples were sequenced on the NovaSeq platform (Illumina, San Diego CA).