title: GSM8701352: untreated; Mus musculus; RNA-Seq
library_construction_protocol: To isolate HSCs, we used wild-type female C57BL/6 mice over 24 weeks old with the Nycodenz density gradient centrifugation method. Vitamin A-containing granules are increased in HSCs along with aging, enabling sufficient numbers of HSCs for qPCR. Briefly, after perfusing livers with 0.3 mg/mL collagenase for 8 min, the livers were perfused with LPB2 (136 mM NaCl, 5.4 mM KCl, 5 mM CaCl2, 0.5 mM NaH2PO3 2H2O, 0.42 mM Na2HPO3, 10 mM HEPES pH 7.5, 5 mM glucose and 4.2 mM NaHCO3) containing 0.6 mg/mL pronase E (KA-002, KNF) and 0.06 mg/ml DNase I at a flow rate of 3 ml/min for 8 min. The perfused livers were transferred to a glass dish and dispersed with tweezers in LPB2 containing 0.6 mg/ml of pronase and collagenase. The cell suspension was transferred to a beaker and incubated at 37°C for 30 min with occasional stirring. Cells were passed through a 70 mm cell strainer and centrifugated at 600 × g for 10 min. The pellet was resuspended in GBSS/B (136 mM NaCl, 5 mM KCl, 1 mM MgCl2 6H2O, 0.28 mM MgSO4 7H2O, 0.22 mM KH2PO4, 0.42 mM Na2HPO4 12H2O, 5.5 mM glucose, 2.7 mM NaHCO3, 1.5 mM CaCl2 2H2O) containing 0.06 mg/ml DNase I, and centrifuged at 600 × g for 10 min two times. The pellets were resuspended in GBSS/B containing 8.8% Nycodenz (18003, SEW), and GBSS/B was gently added to the cell suspension. After centrifugation at 1500 × g for 22 min without braking, HSCs were identified as a condensed white band at the interphase. The HSC fractions were collected with a Pasteur pipette and centrifuged at 600 × g for 10 min. Typically, 1.0 × 106 cells from two mice. Approximately 80-90% of cells were UV-positive by confocal microscopy. Total RNA was extracted from HSCs using RNeasy Micro Kit (74004, QIAGEN). RNA libraries were prepared using the SMART-Seq HT PLUS Kit (R400748, Takara Bio) according to the manufacturer's protocol. Briefly, first-strand cDNA was synthesized using SMARTScribe Reverse Transcriptase with template switching activity at the 5' end of the RNA template. After PCR amplification and purification of double-stranded cDNA, the libraries were enzymatically fragmented and ligated with stem-loop adapters. The libraries were then amplified and indexed with unique dual indexes. Library quality and quantity were assessed using an Agilent 2100 Bioanalyzer with High Sensitivity DNA Kit (Agilent Technologies). Paired-end sequencing (2 × 150 bp) was performed on a NovaSeq 6000 system (Illumina) to generate approximately 40M reads (20M pairs) per sample, with a target of 6G bases per sample.
identifiers.namespace: GEO
identifiers.id: GSM8701352_r1