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Sample GSM7903050

Query DataSets for GSM7903050

Status

Public on May 20, 2024

Title

MAC-T cells, IL10Rα knockout, MAP infection, rep3 (KO-MAP_15)

Sample type

SRA

Source name

mammary epithelium

Organism

Bos taurus

Characteristics

tissue: mammary epithelium
genotype: IL10R{alpha} knockout
treatment: MAP infection

Treatment protocol

The IL10RA KO and Wild type MAC-T cells were seeded at 1.2 x10^5 cells per well in separate 24-well plates and incubated overnight at 37°C and 5% CO2 to reach 80% confluence. Both cell types were either infected with MAP or provided an equivalent volume of MAP-carrier solution media (uninfected control) for 72 h. Bacterial CFU were determined using the pellet wet-weight method, whereby 1 mg of MAP Madonna pellet was equal to 10^7 CFU. MAP was added to each cell type to achieve a 10:1 multiplicity of infection and then spun for 2 min at 250x g to ensure MAP interaction with the cells.

Growth protocol

MAC-T wild type and IL10RA KO cell line were cultured in T25 tissue culture flasks (Corning, Tewksbury, MA, USA) at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Burlington, ON, Canada) supplemented with 4.0 mM L-glutamine, 10% heat inactivated fetal bovine serum (FBS; Invitrogen), 25 mM HEPES buffer (Invitrogen), 0.25 μg/ml amphotericin B (Invitrogen), 1% Penicillin/Streptomycin (100 unit/ml of Penicillin and 100 μg/ml Streptomycin; Invitrogen), 1 mM Sodium Pyruvate (Invitrogen), and 5 μg/ml insulin-transferrin-selenium (Invitrogen).
Mycobacterium avium subsp. Paratuberculosis (MAP) was cultured in Middlebrook 7H9 broth (Sigma-Aldrich) supplemented with 10% oleic acid, albumin, dextrose, catalase (OADC; Becton, Dickinson, Canada), 0.05% Tween 80 (Sigma-Aldrich), and 2 mg/L mycobactin J (Allied Monitor Inc., Fayette, MO). The bacterial culture was incubated at 37°C and 5% CO2.

Extracted molecule

total RNA

Extraction protocol

Total RNA extraction was carried out using the RNeasy mini kit (Qiagen, Germany) from both uninfected and MAP-infected samples according to the manufacturer’s protocol. The DNA traces were removed by DNase I treatment (Fermentas, Waltham, MA, USA). The concentration and purity of the RNA samples were determined at A260/280 nm ratios using the Cytation-5 Spectrophotometer. The integrity of the RNA samples was assessed using Bioanalyzer. The samples with higher than 7 RNA Integrity Number (RIN) were chosen for RNA Seq.
RNA-seq, including library construction were outsourced to Genewiz, Azenta Life Sciences, US

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

CLC Genomics Workbench software 20.0.4 (QIAGEN, Aarhus, Denmark)
The sequence data were trimmed for adaptor sequence, low-quality sequence (Phred score >30) using CLC genomic benchwork (parameter- Quality limit: 0.05 and max 2 ambiguous nucleotides)
The trimmed pair-end sequence reads were mapped to the bovine reference genome (ARS-UCD1.2) using CLC genomic benchwork (parameters- mismatch cost: 2 insertion cost: 3 deletion cost: 3 length fraction: 0.6 similarity fraction: 0.6)
Differentially expressed (DE) genes analyses for "MAC-T cells, wildtype, no infection" versus "MAC-T cells, wildtype, MAP infection", "MAC-T cells, wildtype, no infection" versus "MAC-T cells, IL10Rα knockout, no infection", "MAC-T cells, wildtype, MAP infection" versus "MAC-T cells, IL10Rα knockout, MAP infection", and "MAC-T cells, IL10Rα knockout, no infection" versus "MAC-T cells, IL10Rα knockout, MAP infection" contrasts were performed
Gene expression levels were quantified in reads per kilobase per million mapped reads (RPKM) and transformed to log 2 were performed using CLC genomic benchwork
DE genes between each contrast were defined by a p-value ≤0.01, FDR ≤0.05, and |fold change|≥2.
Assembly: Bos taurus ARS-UCD1.2
Supplementary files format and content: Common delimited text files. Chromosome, start and end physical position, fold change, p-value, FDR p-value correction, gene, Ensemble ID, and RPKM (reads per kilobase of transcript, per million mapped reads) for each of the samples being included in the contrast.

Submission date

Nov 15, 2023

Last update date

May 20, 2024

Contact name

Christine Francoise Baes

E-mail(s)

cbaes@uoguelph.ca

Organization name

University of Guelph

Department

Animal Biosciences