title: GSM8438160: Zona Incerta single nuclei, rep 1; Mus musculus; RNA-Seq
library_construction_protocol: Mice were deeply anesthetized during the light phase of the cycle with saturated isofl-rane vapor and then rapidly decapitated. Each head was submerged in ice cold high sucrose, cutting artificial cerebrospinal fluid (ACSF; 194mM Sucrose, 30mM NaCl, 4.5mM KCl, 1.2mM NaH2PO4, 26 mM NaHCO3, 10mM D-(+)-Glucose, 8mM MgSO4, 0.2mM CaCl; 360 mOsm) saturated with 95% O2 and 5% CO2 and incubated for 1 minute prior to brain dissection and tis-sue slice preparation. 250µm sagittal slices spanning the mediolateral extent of the zona incerta (ZI) were prepared on a vibratome (Leica VT1200S) and the ZI was micro-dissected under ste-reoscopic guidance with the slices submerged in the same ice-cold high sucrose ACSF. Reflected light helped to identify anatomical landmarks (e.g. medial lemniscus, cerebral peduncle) that partially demarcated the boundaries of the ZI. The dissected tissue was then immediately submerged in room temperature RNAlater solution (Thermo Fisher Scientific, AM7020), before it was transferred to 4˚C overnight, and then frozen at -80˚C until further processing. Nuclei were isolated from RNAlater preserved tissues using a previously published method (Habib et al., 2017), except that the final suspension buffer was replaced with a blocking buffer consisting of 1x PBS, 0.5% BSA (Sigma, Cat# B8667-5ML) and 0.2 U/µl RNase inhibitor (Clontech, Cat #2313A) for the neuronal nuclei enrichment process described as follows. The nuclear pellet was resuspended in 600 µl blocking buffer and incubated on ice for 15 minutes followed by add-ing 1.2 µl Anti-NeuN Antibody (clone A60, Alexa Fluor®488 conjugated, EMD Millipore, Cat# MAB377X) and incubation on ice for another 15 minutes. The nuclei suspension was spun down at 500g and 4°C for 5 minutes followed by supernatant removal. The nuclear pellet was resus-pended in 500 µl resuspension buffer consisting of 1x PBS, 1% BSA (Sigma, Cat# B8667-5ML) and 0.2 U/µl RNase inhibitor (Clontech, Cat #2313A) followed by filtration through a 20 µm filter (Sysmex, Cat# 04-004-2325. 1 µl of Vybrant DyeCycle Violet nuclei stain (Invitrogen, Cat# V35003) was added before a SH800 cell sorter (SONY Biotechnology) for FANS by gating on forward and side scatter plot, and both Violet+ NeuN+ to collect the neuronal nuclei population. Nuclei from each tissue sample were loaded (the number of nuclei per sample depended on the sorting yields) onto each channel of the 10x Chromium Chip B (10x Genomics, 1000073) followed by construction of  snRNA-seq libraries with the Chromium Single Cell 3' GEM, Library & Gel Bead v3 Kit (10x Genomics, 1000075) following vendor protocols. The final libraries were pooled and sequenced with a NextSeq500 75 cycle flow cell (Illumina, 20024906) with read length as 28 bases for read1, 55 bases for read2 and 8 bases for index1. snRNA-seq