title: GSM7528322: LC2; Mus musculus; RNA-Seq
library_construction_protocol: Three- to 4-week-old C57Bl/N mice of both sexes were used for cell collection and capturing as described. Briefly, mice were deeply anesthetized and perfused with 40 ml of ice-cold preoxygenated (95% O2/5% CO2) solution containing the following (in mM): 93 N-methyl-d-glucamin-HCl, 30 NaHCO3, 2.5 KCl, 1.2 NaH2PO4, 20 HEPES-NaOH, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2, 8 MgSO4, and 25 glucose (pH 7.4). Next, brains were rapidly extracted and immersed in the same solution to cut 250-μm-thick coronal slices spanning the LC on a vibratome (VT1200S, Leica). After manual isolation of the LC under a stereomicroscope, tissue blocks were subjected to cell dissociation using the papain dissociation system (Worthington) according to the manufacturer's recommendations. Single cells were re-suspended in sterile cutting solution supplemented with 0.1% BSA, fixed in ice-cold methanol for 10 min, and stored at -80 °C until library preparation. For single-nucleus RNA-seq, nuclei were isolated from snap-frozen tissues by direct dissociation in cold Nuclei EZ Lysis buffer (Sigma # NUC101) using a pestle. The lysate was incubated on ice for 10 min, filtered through 70 µm-strainer meshes, and centrifuged at 500 rcf at 4˚C for 5 min. Pelleted nuclei were washed in 'nuclei wash and resuspension buffer' [1% BSA in 1x DPBS plus 0.5 U/µl Protector RNase-Inhibitor (Sigma #3335402001)], followed by centrifugation at 500 rcf at 4˚C for 5 min. Pelleted nuclei were then washed 3x before taking it up in nuclei wash and resuspension buffer supplemented with 7-amino-actinomycin D (7-AAD, BioLegend # 420404). After straining through a 40-µm mesh, single nuclei were sorted using a SONY SH800S cell sorter. Sorted nuclei were processed for single nuclei RNA-seq using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10x Genomics #PN1000128) according to manufactures instructions.