Single-cell RNA-sequencing of the mouse Dorsomedial Hypothalamic Nucleus (DMH)
Single-cell RNA-seq data from the mouse Dorsomedial Hypothalamic Nucleus (DMH) was generated following a modified protocol from https://www.nature.com/articles/s41586-020-2821-8. Briefly, six 7.5 week old C57B6 mice (3 male, 3 female) were anaesthetized with isoflurane. Brains were rapidly extracted and dropped into ice-cold carbogenated NMDG-HEPES-ACSF. Brain sections (2 mm) containing DMH were cut with a razor blade on a stainless steel brain matrix (51392, Stoelting) and transferred to a dissection dish  on  ice  containing  NMDG-HEPES-ACSF.  DMH  was  bilaterally  microdissected  in  ~700·700·350 micron tissue block. Microdissected tissue was aggregated in a collection tube on ice containing NMDG-HEPES-ACSF  and  30  µM  actinomycin  D.  For  enzymatic  tissue  digestion,  NMDG-HEPES-ACSF  was replaced  by trehalose-HEPES-ACSF  containing papain  (50  U/ml;  P3125,  Sigma Aldrich,  pre-activated with 2.5 mM cysteine and a 30-min incubation at 34 °C and supplemented with 0.5 mM EDTA) and 15 µM actinomycin D. Extracted DMH tissue was incubated at room temperature with gentle carbogenation for 55 min. During enzymatic digestion the tissue was pipetted periodically every 10 min. At the end of enzymatic digestion, the medium was replaced with 200µl of room temperature trehalose-HEPES-ACSF containing  3  mg/ml  ovomucoid  inhibitor  (OI-BSA,Worthington)25  U/ml  DNase  I (90083,  Thermo Scientific) and 15 µM actinomycin D and tissue was gently triturated into a uniform single-cell suspension with fire-polished glass Pasteur pipettes. The resulting cell suspension volume was brought up to 1 ml with trehalose-HEPES-ACSF with 3 mg/ml ovomucoid inhibitor and pipetted through a 40µm cell strainer. Single-cell suspension was centrifuged down at 300g for 5 min at 4 °C and the supernatant was replaced with 1 ml fresh ice-cold trehalose-HEPES-ACSF and the cell pellet was resuspended. Cells were pelleted again  and  resuspended  in  100  µl  of  ice-cold  resuspension-ACSF.  Cell  suspensions  were  kept on  ice while cell densities were quantified with a haemocytometer and the final cell densities were verified to be in  the  range  of  300–1,000  cells/µl.  Cell  suspension  volumes  estimated  to  retrieve  ~10,000  single-cell transcriptomes were added to the 10x Genomics RT reaction mix and loaded to the 10X Next GEM Chip G  (2000177, 10x Genomics) per the manufacturer's protocol. We used the Chromium Next GEM Single Cell 3' Reagents Kit v3.1 (1000128, 10x Genomics) and the Single Index Kit Set A (100213) to prepare Illumina sequencing libraries downstream of reverse transcription following the manufacturer's protocol. Resulting  scRNA-seq  sequencing  library  was  sequenced  on  an  Illumina  NovaSeq  6000  sequencers (paired-end 150). Sequencing  data  were  aligned  to  optimized  pre-mRNA  reference  transcriptome52  and  digital  gene-cell matrices were generated with the 10x Genomics Cell Ranger v.6.0.0 count pipeline. The resulting scRNA-seq  data  were  analyzed  in  R  (4.1.2)  using  Seurat  (v.4.1.0.9007)  as  previously  described51. Briefly, expression data were filtered to exclude cells with fewer than 1,000 unique transcripts as well as cells exhibiting  more  than  15%  of  mitochondrial  transcripts.  We  followed  the  standard  Seurat  workflow  to identify transcriptomic cell-types and assigned cel class identities based on canonical cell-type markers: Ndrg4 for neurons, Ntsr2 for astrocytes, Slco1c1 for endothelial cells, Fcer1g for microglia and Mag for oligodendrocytes. We digitally extracted neuronal clusters from our dataset resulting in expression data for 4587 DMH neurons that we used to establish an unbiased neuronal nomenclature for DMH. Overall design: Two separate groups of mice (3 male, 3 female each per sample) were processed for tissue extraction from DMH and single cell suspension generation.