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Sample GSM4474615

Query DataSets for GSM4474615

Status

Public on Apr 14, 2020

Title

Dog 5: Canine primary sublingual epithelial cells, 20 µg/ml D. farinae extract (D5C2)

Sample type

SRA

Source name

Epithelial cells isolated from the sublingual mucosa

Organism

Canis lupus familiaris

Characteristics

sample id: Dog 5
tissue: Epithelial cells isolated from the sublingual mucosa
treatment: 20 ug/ml D. farinae extract

Treatment protocol

Canine primary sublingual epithelial cells were seeded in SECCM in a 24-well plate at 30,000 cells/well and incubated overnight at 37°C and 5% CO2. Thereafter, Dermatophagoides farinae extract, TLR ligands, calcitriol or control medium was added for 24h to the cultured cells, the supernatant was collected and stored at -20°C. The products, listed in Table 1, were added in a volume of 400 µl sublingual epithelial cell stimulation medium (SECSM) (¾ DMEM, ¼ Ham’s F-12, 100 U/ml penicillin, 100 µg/ml streptomycin and 100 µg/ml gentamicin). Since calcitriol was dissolved in ethanol, the control wells for calcitriol contained an equal volume of this solvent. To analyze the effect of D. farinae extract in combination with calcitriol, cells were incubated without stimulus, with 20 µg/ml D. farinae extract or with both D. farinae extract and 0.1 µM in SECSM. Additionally, after 6h incubation with 0.1 µM calcitriol and 20 µg/ml D. farinae extract, sublingual epithelial cells were lysed in RLT buffer (RNeasy mini kit, Qiagen, Hilden, Germany) and stored at -80°C.

Growth protocol

Sublingual biopsies of approximately 0.5x0.5 cm were sampled within 3 hours after euthanasia from 6 privately-owned dogs that were euthanized for medical reasons unrelated to the study (Supplementary Table A). Oral consent to use the corpse for scientific purposes was given by all dog owners. Of one dog, one half of the sublingual biopsy was processed for immunohistochemistry. This sample was submerged in Methocel® MC (Merck), snap-frozen in liquid nitrogen and stored at -80°C. To isolate sublingual cells, biopsies were briefly submerged in 70% ethanol, washed 3 times with Ca2+- and Mg2+-free Dulbecco’s PBS (DPBS, Thermofisher Scientific, Waltham, MA, USA) and incubated in 4 mg/ml Dispase II (Thermofisher Scientific) in DPBS on ice. After 16-24h incubation, the epithelial layer was carefully separated from the underlying subepithelial tissue, followed by incubation of the epithelial and subepithelial tissue in DPBS with 0.25% trypsin (Thermofisher Scientific) and 2.65 mM EDTA for 20 min at room temperature (RT; 18-22°C). Subsequently, cell suspensions were resuspended, trypsin was neutralized with an equal volume of 5% fetal calf serum (FCS) (Merck, Burlington, MA, USA) in DPBS and the cells were filtered over a 70 µm cell strainer (Merck). After centrifugation (400g, 10 min, 18°C), cells were counted and 2.5x105 cells/cm2 were seeded in a culture flask in sublingual epithelial cell culture medium (SECCM; ¾ DMEM, ¼ nutrient mixture F12-Ham (Thermofisher Scientific), 5% FCS, 2 nM 3,3’,5-triiodo-L-thyronine sodium salt, 5 µg/ml recombinant human insulin, 10 ng/ml recombinant human epidermal growth factor, 0.4 µg/ml hydrocortisone, 100 nM L-isoproterenol hydrochloride (Merck), 100 U/ml penicillin, 100 µg/ml streptomycin and 100 µg/ml gentamicin (Thermofisher Scientific)) (27,28). During the first 7 days of culture, the SECCM was supplemented with 2.5 µg/ml amphotericin B (Thermofisher Scientific) and medium was changed daily. Thereafter, medium was replaced every 2-3 days. Cells were expanded, split (0.25% trypsin, 1 mM EDTA in DPBS) at 70-90% confluency and used at passage 2-7 for the experiments. The sublingual epithelial cells were shown to be free of Mycoplasma infection during routine testing using the LookOut® Mycoplasma PCR Detection Kit (Merck) according to the manufacturer’s instructions.

Extracted molecule

total RNA

Extraction protocol

RNA extraction and DNase treatment of the samples was performed using the RNeasy mini kit (Qiagen) and RNase-Free DNase Set (Qiagen) according to the manufacturer’s instructions. Extracted RNA was stored at -80°C.
Total RNA concentration was measured using the Quant-iT RiboGreen RNA Assay Kit (Thermofisher Scientific). RNA quality was inspected using an RNA 6000 Nano chip on a BioAnalyser (Agilent Technologies, Santa Clara, CA, USA). For each sample, a sequencing library was constructed using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) starting from 500 ng total RNA and amplified by 14 PCR cycles. Quality and size distribution of the libraries were checked on a BioAnalyser (Agilent Technologies) using the high sensitivity Quant-iT dsDNA Assay Kit (Thermofisher Scientific) and a High Sensitivity DNA chip. Library concentration was determined according to Illumina’s Sequencing Library qPCR Quantification Guide on a LightCycler 480 (Roche Life Science, Penzberg, Germany).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Sequencing read quantity and quality was checked using FastQC (v0.11.8). Contamination was determined using FastQ Screen (v0.14.0). Adapter trimming and removal of reads having a quality score lower than 20 or containing any ambiguities, was done using cutadapt (v2.5). The remaining high-quality reads were mapped on the CanFam3.1 reference dog genome using STAR (v2.7.2) and features were count at the gene level using RSEM (v1.3.1).
Differential expression analyses of the RNA-sequencing data were done in R using the edgeR (v3.26.8) package. For each analysis, feature counts were normalized using edgeR’s standard normalization method.
To remove uninformative low-count features, those having less than 1 cpm (counts per million) in the number of samples equivalent to the size of the smallest group were discarded.
Statistical testing was done by fitting a general linear model, followed by a quasi-likelihood F-test.
Differentially expressed features were considered statistically significant when having a fold-change of at least 2 (doubling or halving between the compared groups) and a false discovery rate (FDR or adjusted p-value according to Benjamini-Hochberg) smaller than or equal to 0.05.
Genome_build: CanFam3.1 reference dog genome
Supplementary_files_format_and_content: xlsx

Submission date

Apr 13, 2020

Last update date

Apr 15, 2020

Contact name

Michael Pelst

Organization name

Ghent University

Street address

Salisburylaan 133

City

Merelbeke