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Sample GSM6032753 Query DataSets for GSM6032753
Status Public on May 09, 2023
Title CTR_24h_m2
Sample type SRA
 
Source name Liver
Organism Mus musculus
Characteristics strain: C57BL/6
gender: Male
animal id: m2
tissue: Liver
treatment: CTR
timepoint: 24h
Treatment protocol Glucose_24h_25mM
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).
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).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description 1048_0017
1048_0017
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).
 
Submission date Apr 07, 2022
Last update date May 09, 2023
Contact name Eric Simon
E-mail(s) eric.simon@boehringer-ingelheim.com
Organization name Boehringer Ingelheim Pharma GmbH & Co KG
Street address Birkendorfer Str. 65
City Biberach a.d. Riss
ZIP/Postal code 88397
Country Germany
 
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
Relations
BioSample SAMN27404568
SRA SRX14777030

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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