GSM7706473: vlPAG nuclus, HFD 7W; Mus musculus; RNA-Seq
C57/BL6J mice (8 weeks old at the start of diet manipulation) were fed with on either NFD (n = 18 mice) or HFD (n = 18 mice) for 7 weeks. Mice were then deeply anesthetized with pentobarbital and sacrificed, followed by transcardial perfusion with ice-cold cutting solution containing (in mM): 110 Choline chloride, 2.5 KCl, 1.3 NaH2PO4, 7 MgCl2·6H2O, 0.5 CaCl2·2H2O, 25 NaHCO3, 20 D-glucose. Brains were quickly removed, and coronal sections of 300 μm covering the whole vlPAG were obtained using a Leica VT1200S vibratome. Then the sections were placed under a dissecting microscope (Olympus, SZ61) and dissected with surgical instruments for retinal operation. Afterwards tissues were stored at -80℃ until further processing. Nuclei were isolated using Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (5892791001; Roche) and RNase inhibitor (N2615; Promega and AM2696; Life Technologies). Single-nuclei suspensions were loaded to 10X Chromium to capture 8000 single cells using a 10X Genomics Chromium Single-Cell 3' kit (V3) according to the manufacturer's instructions.The following cDNA amplification and library construction steps were performed according to the standard protocol. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run, 150 bp) by LC-Bio Technology co.ltd., (HangZhou, China).