GSM8967903: PVN cells, saline group 2; Mus musculus; RNA-Seq
For optimal nuclei recovery, PVN was microdissected from control mice (n = 6/sample, 2 samples/group) 3 h post-modeling and were immediately placed in ice-cold PBSE buffer (1× PBS containing 2 mM EGTA). Tissue dissociation was performed using gentle mechanical homogenization followed by enzymatic treatment when necessary. Nuclei isolation was carried out using the GEXSCOPE® Nucleus Isolation Kit (Singleron Biotechnologies), with all steps performed on ice to maintain RNA integrity. The homogenate was sequentially filtered through 100 μm and 40 μm cell strainers to remove debris and obtain single nuclei suspensions. Nuclei viability and concentration were assessed using DAPI staining (1:1000 dilution) and automated cell counting with Trypan Blue exclusion. Only intact, single DAPI-positive nuclei with clear nuclear morphology were selected for downstream processing, with final concentrations adjusted to 1×10⁶ nuclei per 400 μL in nuclei preservation buffer. The concentration of singlenucleus suspension was adjusted to 3-4 × 105 nuclei/mL in PBS. Single nucleus suspension was then loaded onto a microfluidic chip (GEXSCOPE® Single NucleusRNA-seq Kit, Singleron Biotechnologies) and snRNA-seq libraries were constructed according to the manufacturer's instructions (Singleron Biotechnologies). The resulting snRNA-seq libraries were sequenced on an Illumina novaseq 6000 instrument with 150 bp paired end reads.