title: GSM6500695: SCN, mixed, rep 2 [SCN2]; Mus musculus; RNA-Seq
library_construction_protocol: Mice were sacrificed and brains were immediately extracted and dropped into ice cold Hanks' Balanced Salt Solution (HBSS). After 2 minutes, brains were embedded in low melting point agarose (Precisionary Instruments, Natick, MA) and sectioned at 400 um on a Compresstome VF-200 Vibrating Microtome (Precisionary Instruments, Natick, MA, USA) into DNAse/RNAse free 1x PBS. Hypothalamic sections of interest were immediately collected into RNAprotect (Qiagen, Hilden, Germany) and kept in RNAprotect at 4oC overnight. The next day Lepr-Cre::TdTomato positive cells were visualized using a fluorescent stereoscope (Leica, Wetzlar, Germany). tdTomato fluorescence was used to approximate DMH and SCN boundaries during microdissection of these regions. DMH and SCN microdissected tissue samples were placed into Eppendorf tubes separated by brain region and feeding condition, and stored in -80C until nuclei isolation. DMH and SCN microdissected tissue samples were transferred from a -80oC freezer and into individual 2mL glass dounce homogenizer tubes (Kimble, Vineland, NJ, USA) to be homogenized according to a protocol modified from Habib et al., 2016 (PMID: 27471252). Tissue was homogenized in 1mL of buffer (16mM sucrose, 5mM CaCl, 3mM Mg(Ac)2, 10mM Tris pH 7.8, 0.1mM EDTA, 1% NP40, 1mM beta-mercaptoethanol in H2O) on ice, with 25 passes of pestle A and 25 passes of pestle B. An additional 4mL of buffer was added to the nuclei suspension and placed on ice for 5 minutes. Then 5 mL of 50% OptiPrep Density Gradient Medium [Sigma Aldrich, MO, USA] (30mM CaCl, 18mM Mg(Ac)2, 60mM Tris pH 7.8, 0.1 mM PMSF, 6mM beta-mercaptoethanol in H2O; in 60% (w/v) solution of iodixanol in sterile water) was added to nuclei on ice and inverted 10 times to mix. The nuclei suspension was layered onto 10 mL of 29% OptiPrep solution (Buffer + 50% OptiPrep) in a 38.5 mL Ultra-Clear tube (Beckman-Coulter, CA, USA) before being centrifuged at 7,333g for 30 minutes at 4oC. Supernatant was discarded and the nuclei pellet was resuspended in 1x PBS + 1% BSA (Sigma-Aldrich, MO, USA) + 2 mM Mg2+ + 0.1% RNase inhibitor (Sigma-Aldrich, MO, USA) for 15 minutes on ice. The nuclei suspension was pipetted through a 20 micron mesh filter along with 2 drops of propidium iodide (PI) Ready Flow reagent (Thermo Fisher, MA, USA) and immediately taken on ice to be FACS sorted using an SH800 (Sony, Tokyo, Japan) cell sorter. Sorting was gated to select for PI+ single nuclei. Nuclei were sorted through a 70 micron nozzle into a 2mL LoBind collection tube (VWR, PA, USA) containing 18.8uL of RT Reagent B from the Chromium Next GEM Single Cell 3' Reagent Kit v3.1. The remaining components of the 10X Step 1 mastermix were then gently mixed with the contents of the FACS collection tube and loaded into the 10X Genomics Chromium Controller Chip G. The single-nuclei samples were processed into sequencing libraries using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 according to the manufacturer's protocol (version 3.1, revision D). After generation of the GEMs (Gel Bead-In EMulsions) and reverse transcription of poly-adenylated mRNA, cDNA was amplified (10-14 cycles), enzymatically fragmented, and ligated to Illumina adapters. Sequencing libraries were indexed, size selected for 400-600bp using SPRIselect (Beckman Coulter, Indianapolis, IN, USA), and quantified by Qubit (v4.0, 1X high-sensitivity dsDNA kit, Thermo Scientific) and Bioanalyzer (Agilent, Santa Clara, CA, USA). Size-corrected library concentrations were used to pool libraries for equimolar representation. The pooled concentration was measured by KAPA Library Quant qPCR according to the manufacturer's instructions (KAPA Biosciences, Wilmington, MA) and by Qubit. Library pools were sequenced using a P100 cycle kit on the NextSeq 2000 (Illumina, CA, USA) in the University of Virginia School of Medicine Genome Analysis and Technology Core, RRID:SCR_018883. The sequencing structure was as follows: Read 1 was 28 bp (16 bp barcode, 12 bp UMI), Read 2 was 98 bp (cDNA) and Index 1 was 8 bp (single index). Overall, we had 7 DMH and 5 SCN sequencing library pools. 10X Batch 1 & 2 contained mixed pools of all feeding conditions, but 10X Batch 3 contained a unique feeding condition per library pool snRNA-seq using Chromium Next GEM Single Cell 3' Reagent Kit v3.1