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Status |
Public on Apr 17, 2024 |
Title |
ctrl-LP24 |
Sample type |
SRA |
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Source name |
gastric tissue
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Organism |
Mus musculus |
Characteristics |
tissue: gastric tissue genotype: Trif KO treatment: Uninfected (control)
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Treatment protocol |
This study used a well-characterized cancer mouse model, infecting C57BL/6 mice with H. felis (strain CS1), a close relative of the human gastric pathogen H. pylori. Mice were inoculated with 109 organisms in 300 μL of BHI by oral gavage repeated three times at 2-day intervals, as previously described. Control mice received BHI only. At 1 month, 3 months, and 6 months post-infection, mice were euthanized, and their stomachs removed under aseptic conditions. The stomach tissue was cut longitudinally, and sections were processed for RNA extraction and histopathology. All H. felis-infected mice were confirmed to be colonized by assessing the expression of the flagella filament B (flab) gene using real-time PCR as described in our previous studies.
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Growth protocol |
Six- to ten- week-old wild type (WT), MyD88 deficient (Myd88-/-), TRIF deficient (TrifLps2), double knockout (Myd88-/- and TrifLps2, DKO) mice in the C57BL/6 background were used in this study. WT mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Myd88-/- mice were from our breeding colony originally provided by Dr. Akira (Osaka University, Japan) and backcrossed 10 times onto a C57BL/6 background, bred, and maintained at our facility. The DKO mice were bred in our facility by crossing Myd88-/- and TrifLps2. All mice were housed together before and throughout the H. felis infection study for each genotype. The animal procedures were approved by the Institutional Animal Care and Use Committee at the University of California, San Diego, and conducted following accepted veterinary standards. Helicobacter felis, strain CS1 (ATCC 49179) was obtained from the American Type Culture collection (Manassas, VA). The bacteria were cultured on solid Columbia agar (Becton Dickinson, MD) supplemented with 5% laked blood under microaerophilic conditions (5% O2, 10% CO2, 85% N2) at 37 °C and passaged every 2–3 days, following established protocols (7, 16-18). Prior to mouse infections, H. felis was cultured in liquid brain heart infusion broth (BHI, Becton Dickinson) supplemented with 10% fetal calf serum and incubated at 37 °C under microaerophilic conditions for 48 h. Spiral bacteria were enumerated using a Petroff-Hausser chamber before infections.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted from gastric tissue obtained from H. felis-infected and uninfected WT, Myd88-/-, TrifLps, and DKO mice. Gastric tissue samples were homogenized in 1 ml of TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. RNA was extracted using the Direct-zol RNA mini kit (Zymo Research, Irvine CA) as per the manufacturer’s instructions and stored at -70°C until further use. The quality of the extracted RNA was assessed using a Nanodrop system (Thermo Fisher, Waltham MA) by measuring absorbance levels at 260 and 280 nm. Samples were processed into RNA-seq libraries using the Illumina TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero (Human/Mouse/Rat), which involved ribosomal RNA depletion, fragmentation, cDNA synthesis, and cDNA library preparation.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
RNA-seq profiling of gastric tissue from uninfected Trif LPS2 KO mice
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Data processing |
For RNA-seq data analysis, the reads were aligned to the combined mouse and H. felis genomes (GRCm38/mm10 and NC_014810.2, respectively) using STAR with default parameters. Only reads that aligned uniquely to a single location acrossin the genomes with a mapping quality score greater than 10 (MAPQ > 10) were used for downstream analysis. Gene expression levels were determined by counting the number of reads overlapping exons for all genes using HOMER’s analyzeRepeats.pl tool and the transcriptome annotation from GENCODE (version M25). Gene expression values were normalized using DESeq2’s rlog function. Gene expression values for genes on the H. felis genome were determined using HOMER’s analyzeRepeats.pl function based on gene annotations associated with NCBI accession number NC_014810.2, and reported as fragments per kilobase per million uniquely mapped fragments (FPKM), normalized to the total number of reads aligning to both the mouse and pathogen genomes. Assembly: GRCm38/mm10, NC_014810.2 Supplementary files format and content: mouse.rlog.txt: Tab delimited text file of DESeq2 rlog normalized (i.e. log2) gene expression values for all mouse GENCODE genes across all samples. Supplementary files format and content: hfelis.fpkm.txt: Tab delimited text file of FPKM normalized (to both host and pathogen read counts) gene expression values for all H. felis genes across all samples.
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Submission date |
Dec 18, 2023 |
Last update date |
Apr 17, 2024 |
Contact name |
Christopher Benner |
E-mail(s) |
cbenner@ucsd.edu
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Organization name |
University of California, San Diego (UCSD)
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Department |
Medicine
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Street address |
9500 Gilman Dr. MC 0640
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City |
La Jolla |
State/province |
California |
ZIP/Postal code |
92093-0640 |
Country |
USA |
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Platform ID |
GPL17021 |
Series (1) |
GSE250438 |
Role of the TRIF-IFN-I pathway in Helicobacter-induced gastric cancer progression in an accelerated murine disease model |
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Relations |
BioSample |
SAMN38882209 |
SRA |
SRX22920890 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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