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| Status |
Public on May 09, 2023 |
| Title |
GFIO_48h_m1 |
| Sample type |
SRA |
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| Source name |
Liver
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
gender: Male
animal id: m1
tissue: Liver
treatment: GFIO
timepoint: 48h
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| Treatment protocol |
Glucose_48h_36mM_Fructose_48h_Insulin_48h_Oleic Acid_48h
Biopsies (6 mm diameter) were made from liver tissues, and sliced using a Krumdieck tissue slicer (Alabama Research and Development, Munford, USA) according to the protocol described by de Graaf et al (Nature protocols paper). Precision-cut liver slices (PCLSs) had a wet weight of ~5 mg. PCLSs were cultured at 37degC under 80% O2 and 5% CO2, as previously described9. Culture lasted 24 or 48 hours and the culture medium was refreshed every 24 hours. Different culture media were used to mimic metabolic abnormalities. The control medium consisted of Williams medium E with Glutamax (Invitrogen, Bleiswijk, The Netherlands), 50mg/mL gentamycin (Invitrogen), and 25mM glucose (Merck, Darmstadt, Germany). Hyperglycemia was simulated by addition of 36mM glucose or 5mM fructose (Merck).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Three PCLS slides per donor per assay condition have been pooled to ensure to get enough material for RNA Sequencing. After homogenization and lysis using in Qiazol buffer (Qiagen, Venlo, the Netherlands), samples were mixed with 1/5th volume of chloroform (Sigma) and separated using phase extraction tubes. The RNA was then isolated with the RNeasy Lipid Tissue Mini Kit (Qiagen, Venlo, the Netherlands) or the FavorPrep TM Tissue Total RNA Mini Kit (Favorgen, Vienna, Austria). RNA samples were quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (ThermoFisher) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 96-channel Fragment Analyzer (Agilent), respectively.
Total RNA was normalized on the MicroLab STAR automated liquid platform (Hamilton). 100ng input was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760, together with the QIAseq FastSelect RNA removal Kit (Qiagen) upstream and the NEBNext Multiplex Oligos for Illumina #E7600 downstream (all New England Biolabs). Fragmentation time was set to 15min. A total of 12 PCR cycles was used for the index PCR while the final libraries were eluted in 25 µL. Of note, the only deviation from the manufacturer’s protocol was the use of Ampure XP beads (Beckman Coulter) at the double-stranded cDNA purification step, instead of the recommended SPRIselect Beads. Total RNA sequencing libraries were quantified by the fluorescence dye-based methodology High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). Library molarity averaged at 100.3 nM. Total RNA libraries were also assessed for size distribution and adapter dimer presence by the High Sensitivity NGS Fragment DNF-474 Kit on a 96-channel Fragment Analyzer (Agilent). Sequencing libraries were normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina).
The library pool was subsequently clustered on an S2 Flow Cell and sequenced on a NovaSeq 6000 Sequencing System (Illumina) with dual index, paired-end reads at 2x100 bp length (Read parameters: Rd1: 101, Rd2: 8, Rd3: 8, Rd4: 101) reaching a target depth of approximately 60 million Pass-Filter reads per sample (6.5% CV, see).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
1048_0015
1048_0015
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| Data processing |
reads passing quality control filter were mapped against or GRCm38 (mouse data) reference genomes using STAR (v2.5.2b)
Gene expression levels were quantified based on genome annotation files from Ensembl version 86 using RSEM (v1.3.0) and featureCounts (v1.5.1). For additional quality control we used FastQC (v0.11.5), picardmetrics (v0.2.4) and dupRadar (v1.0.0).
Assembly: GRCm38 (mouse data)
Supplementary files format and content: 1048_uReads.txt containts tab delimited normalized read counts per gene (row) for all samples (column).
Supplementary files format and content: 1048_uTPM.txt containts tab delimited normalized TPM per gene (row) for all samples (column).
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| Submission date |
Apr 07, 2022 |
| Last update date |
May 09, 2023 |
| Contact name |
Eric Simon |
| E-mail(s) |
eric.simon@boehringer-ingelheim.com
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| Organization name |
Boehringer Ingelheim Pharma GmbH & Co KG
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| Street address |
Birkendorfer Str. 65
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| City |
Biberach a.d. Riss |
| ZIP/Postal code |
88397 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE200408 |
Transcriptomic profiling of induced steatosis in mouse precision-cut liver slices |
| GSE200419 |
Transcriptomic profiling of induced steatosis in precision-cut liver slices |
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| Relations |
| BioSample |
SAMN27404570 |
| SRA |
SRX14777028 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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