title: GSM7324159: Dorsomedial hypothalamus 1; Mus musculus; RNA-Seq
library_construction_protocol: Single-cell RNA-seq data from mouse Dorsomedial Hypothalamus was generated as previously described (https://www.nature.com/articles/s41586-020-2821-8). Briefly, six 7.5-week-old C57BL6/J mice (3 male, 3 female per sample) 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 ~700x700x350 µm tissue block. The 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 trehaloseHEPES-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 hemocytometer 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).