biostudies-arrayexpressShad HassanMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-17544Single-nucleus RNA-seq of mouse mediobasal hypothalamus across dietary states (chow, fasting, refeeding, HFD), leptin IP injection, and Glp1rLeprKO genetic model. Identifies Lepr/Glp1r neurons as a uniquely leptin-sensitive population restraining AgRP-driven hyperphagia in diet-induced obesity.biostudies-arrayexpressNucleic Acid Extraction - Frozen arcuate nucleus samples were pooled in randomized groups of 4-12 across developmental time and group, recovered in ice-cold Nuclei EZ lysis buffer (Sigma #NUC101) and lysed/homogenized with a 2 ml KIMBLE dounce (pestle B, 10 strokes; Sigma #D8938). After a 5 min incubation, debris and myelin were removed through 40 um cell strainers (Pluriselect #43-10040) and centrifugation (1,000 rcf, 5 min, 4 degC). The pellet was resuspended in a 1:1 mix of EZ lysis buffer and 50% OptiPrep iodixanol to 25% (Sigma #D1556), layered on 29% iodixanol and centrifuged (14,000 rcf, 22 min, 4 degC). Nuclei were resuspended in nuclei buffer (PBS, 1% BSA, 2 mM Mg2+, 40 U/ul Protector RNase inhibitor, Roche #3335399001), incubated 15 min, centrifuged (1,000 rcf, 10 min, 4 degC) and resuspended in 100 ul nuclei buffer. For multiplexing, a unique TotalSeq anti-Nuclear Pore Complex Proteins hashtag antibody (0.5 ug/ul; BioLegend A0451-A0465) was added and incubated 30 min. Nuclei were washed, stained with 0.5 ug/ml DAPI, and single DAPI+ nuclei were sorted on a Sony SH800 cell sorter (70 um Sorting Chip, #LE-C3207) directly into Chromium Next GEM Single Cell 3' v3 or v3.1 reverse transcriptase reagent (10x Genomics), targeting ~28,000 nuclei per reaction.Sequencing - Single-cell libraries of endogenous RNA and hashtag oligos were sequenced on SP and S2 flow cells using the Illumina NovaSeq 6000 platform (Read1 28 bp, Index1 8 bp, Read2 94 bp), to a depth of approximately 40,000 reads per cell for the endogenous RNA library and 10,000 reads per cell for the hashtag oligo libraries.Sample Collection - At sacrifice, mouse brains were dissected and immediately placed in an ice-chilled coronal brain slicer (Zivic Instruments #BSMAS005-1 or ASI Instruments RBM-2000C). A 1-1.5 mm thick coronal section was obtained between the rostral and caudal ends of the Circle of Willis and placed in ice-cold PBS. The mediobasal hypothalamus (arcuate nucleus) from the left and right hemispheres was combined in a tube pre-cooled on dry ice and stored at -80 degC. All dissections were conducted between 8 a.m. and 12 p.m. to avoid transcriptional effects from circadian rhythm and feeding patterns.Library Construction - Single-nucleus RNA-seq libraries were generated using the Chromium Next GEM Single Cell 3' kit, dual index, v3 or v3.1 (10x Genomics). The 10x protocol was followed up to cDNA amplification, where 0.2 uM hashtag oligo (HTO) primers were added to the cDNA amplification master mix. Following SPRI clean-up, the pellet was processed per the standard 10x protocol to generate endogenous mRNA libraries. HTO libraries were recovered from the supernatant with 70 ul SPRI beads (Beckman Coulter #B23317), eluted in EB buffer (Qiagen), and indexed by PCR (50 ul Kapa HiFi HotStart ReadyMix 2X, Roche #07958935001; SI PCR primer + Illumina TruSeq D70x index; 98 degC 2 min, 15x [98 degC 20 s, 64 degC 30 s, 72 degC 20 s], 72 degC 5 min). Libraries were quantified by Qubit Fluorometer (Invitrogen #Q32851) and Agilent TapeStation High Sensitivity D1000 (#5067-5584).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Data processing was carried out in Seurat v4.3.0. For each 10X lane, cells with outlier UMI counts were identified and removed. Raw counts were normalized with the NormalizeData functionMetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsMus musculusShad HassanfalseA uniquely leptin sensitive hypothalamic neuron population limits hyperphagia and weight gain in diet-induced obesitySingle-nucleus RNA-seq of mouse mediobasal hypothalamus across dietary states (chow, fasting, refeeding, HFD), leptin IP injection, and Glp1rLeprKO genetic model. Identifies Lepr/Glp1r neurons as a uniquely leptin-sensitive population restraining AgRP-driven hyperphagia in diet-induced obesity.2026-08-27T00:00:00Z2026-08-27T08:33:52.654Z2026-08-26T09:35:34.431ZE-MTAB-17544ERP204324EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184