title: GSM4058667: mouse liver-304-HFD,RNA-s102796-001-030_HFD.fastq.R1; Mus musculus; RNA-Seq
library_construction_protocol: Food Intake, Body Composition    Food intake and body weight were monitored every three weeks of the study. Total body fat mass was assessed with EchoMRI at the start and in week 9, 15 and 21 of the study as previously described [42].   Plasma Measurements   Blood samples were collected after 5 h fast via tail vein bleeding for isolation of EDTA plasma for evaluation of total cholesterol, triglycerides and insulin as described [17]. Plasma HDL-cholesterol was determined after precipitation as described earlier [43] with enzymatic assays (CHOD-PAP, Roche Diagnostics, Almere, the Netherlands). Non-HDL cholesterol was calculated by subtracting HDL-cholesterol levels from total plasma cholesterol levels. Blood glucose was measured using a hand-held glucometer in tail blood during blood collection (FreeStyle Lite, Abbott, Alameda CA, USA).    Sacrifice and Analysis of Fat and Liver Tissue   All animals were sacrificed under non-fasted conditions by CO2 asphyxiation after 21 weeks of dietary treatment. Blood was collected via cardiac puncture for EDTA-plasma collection. White adipose tissue (WAT) was collected from gonadal and mesenteric WAT, abdominal fat mass, and the subcutaneous fat depot. The liver was weighed and divided into different parts. The medial lobe was fixed in formalin and embedded in paraffin. Development of NAFLD was analyzed in 3-μm liver hematoxylin-eosin stained sections and analyzed by a board-certified pathologist using a wellestablished adapted scoring method for human NAFLD [44] as described [17]. Briefly, the level of total steatosis per mouse was expressed as a percentage of the total liver section affected by microvesicular or macrovesicular steatosis. Similarly, hypertrophy was expressed as the affected percentage of the total liver section. Steatosis and hypertrophy were scored in the liver sections at a 50x magnification. The HE stained liver sections were digitalized with a 20 × magnification (Aperio AT2, Leica Biosystems, Amsterdam, The Netherlands) and used for the representative images.   4-HNE Oxidative Stress-Related Marker Staining and Quantification in Liver Tissue    Oxidative stress-related marker 4-hydroxynonenal (4-HNE) was analyzed in 3-μm paraffin liver sections. After deparaffinization of the liver sections, heat-induced epitope retrieval was performed with a PH 6 citrate buffer (PT link, Dako, Denmark). Subsequently, sections were washed with 0.05% Tween-20 in phosphate-buffered saline (PBS) and blocked for 30 min at room temperature (RT) with 5% normal goat serum in PBS. Staining was performed by overnight incubation at 4 °C with Rabbit anti-4-HNE Michael adducts primary antibody (1:1000 in PBS, ref.393207, Millipore Corporation, Billerica, MA, USA PBS). The next day these sections were washed and blocked for endogenous peroxidase with 3% H2O2 during 20 min, and subsequently incubated for 45 min with a BrightVision poly-HRP-anti-rabbit secondary antibody (1:1 in PBS, Immunologic, Duiven, The Netherlands;) all at RT. Lastly, after washing the sections, Diaminobenzidine (DAB) was used for color development by 5 min incubation and nuclei were stained with hematoxylin for 30 s at RT. The 4-HNE stained liver sections were digitalized with a 20 × magnification (Aperio AT2, Leica Biosystems, Amsterdam, The Netherlands). In these scans 4-HNE positive immunoreactivity was scored in two different liver sections in five nonoverlapping fields per liver section and expressed as amount of 4-HNE positive immunoreactivity structures per mm2.    LC-MS/MS   Sacrifice plasma samples were used to measure L-carnitine, nicotinamide and several acylcarnitine levels. The metabolites were measured with the Metabolon analytical system (Metabolon, Inc., Durham, NC, USA). A non-targeted semiquantitative liquid chromatography– tandem mass spectrometry (LC-MS/MS) platform was applied for the identification of structurally named and unknown molecules [45,46]. All normalized relative ion counts were log transformed, and the remaining data were imputed with the minimum value on a per metabolite basis and reported in relative units (RU). Gene Expression and Pathway Analysis   Total-RNA isolation kit (Bio-connect, Huissen, The Netherlands) was used to extract RNA from snap-frozen livers. The RNA concentration was measured spectrophotometrically using a NanoDrop 1000 (Isogen Life Science, De Meern, The Netherlands). To assess the quality of the isolated RNA, the 2100 Bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands) was used. Next, messenger RNA (mRNA) library was produced for next generation sequencing following the manufacturer′s protocol NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The quality and yield of the PCR products were consistent with the expected size distribution (300–800 base pairs). Clustering and DNA sequencing were performed by GenomeScan BV (Leiden, The Netherlands) using a NextSeq 500 sequencer (Illumina, San Diego, CA, USA). The sequences were directly aligned with the mouse reference genome (GRCm38p4) using the Start 2.5 algorithm with default settings. RNA-seq was performed by GenomeScan B.V. (Leiden, NL) bu using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #7420S/L, New England Biolabs, Ipswich, Massachusetts, USA). Briefly, mRNA was isolated from total RNA using oligo-dt magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed which was used for ligation with sequencing adapters, PCR amplification and quality measurement by Fragment-analyser. Size distribution generaly was a broad peak between 300-500bp. Clustering and DNA sequencing was performed on Illumina NextSeq 500 using 1.6 pM DNA.
attributes.tag: GEO Accession
attributes.value: GSM4058667