GSM8330432: STZ-HFHC, 2nd-1-1-3, replicate 1, sNuc Seq; Mus musculus; RNA-Seq
Nuclei were prepared from approximately 100 mg of frozen mouse liver tissue using the Singulator 100 system (S2 Genomics). Following dissociation, debris were eliminated through a Percoll gradient. The nuclei concentration was then evaluated using a LUNA-FL™ Automated Fluorescence Cell Counter (Logos Biosystems), and nuclei morphology was scrutinized through microscopy. Libraries were generated using the Chromium controller in adherence to the 10× Chromium Next GEM Single Cell 3' v3.1 protocol (CG000315). In summary, cell suspensions were diluted with nuclease-free water to attain a targeted cell count of 10,000. The cell suspension was mixed with a master mix and loaded onto a Chromium Next GEM chip G along with Single Cell 3' v3.1 Gel Beads and Partitioning Oil. Within droplets, RNA transcripts from individual cells were uniquely barcoded and reverse transcribed. The cDNA molecules were pooled, underwent end repair, had a single 'A' base added, and the adapters were then ligated. Following purification and PCR enrichment, the final cDNA library was created. Quantification of the purified libraries was performed using quantitative real-time polymerase chain reaction (qRT-PCR) according to the qPCR Quantification Protocol Guide (KAPA), and their quality was assessed using the Agilent Technologies 4200 TapeStation (Agilent Technologies). The libraries were then sequenced on the HiSeq platform (Illumina), adhering to the read length specifications provided in the user guide.