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| Status |
Public on Dec 31, 2022 |
| Title |
Pig olfactory mucosa cells - 15_CFTR |
| Sample type |
SRA |
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| Source name |
Pig olfactory mucosa cells
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| Organism |
Sus scrofa |
| Characteristics |
tissue: Olfactory neuroepithelium
age: Newborn
genotype: CFTR knock out piglet
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| Treatment protocol |
Olfactory neuroepithelium from CFTR-null and control piglets were dissected included in OCT and immediately frozen on liquid N2. 300 µm sections were made using a cryostat, and kept at -80°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
In a glass dounce tissue grinder previously cooled on ice, 7 tissue slices and 1ml of citric acid-based buffer (Sucrose 0.25 M, Citric Acid 25 mM) were added. Tissue samples were homogenized with 3-5 strokes of loose pestle and incubated on ice for 5 min. After 5 more strokes using a loose pestle, samples were incubated on ice for 5 min, homogenized again with 3 strokes using the loose pestle and 5 strokes using the tight one. The suspension was filtered through a 40 µm cell strainer, and 1ml of citric acid-based buffer was used to wash the containers. After a centrifugation for 5min at 500g at 4°C, the supernatant was carefully removed and the sample was resuspended in 1ml of Wash buffer (Tris-HCl pH 7.4 10 mM, NaCl 10 mM, MgCl2 3mM, BSA 1%, Tween 20 0.1%, DTT 1 mM, RNaseIn® 0.6 U/μL, SuperaseIn® 0.2 U/μL). Sample was filtered through a 5 µm cell strainer and centrifuged for 5min at 500g at 4°C. Nuclei were resuspended in 50-100 µl of diluted nuclei buffer (Nuclei Buffer® 1X (Multiome kit, 10X Genomics), DTT 1 mM, RNaseIn® 0.6U/µl, SuperaseIn® 0.2 U/μL). Nuclei morphology and isolation were controlled with a Floid cell imaging station. After counting with a Countess II FL Automated Cell Counter, nuclei were diluted to the desired concentration (for a target capture of 10,000 nuclei).
snNucSeq was performed according to the protocol provided by 10X Genomics.
snNucSeq (10X Genomics)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Illumina NextSeq500 software used for basescaling
Raw data were analyzed using Cell Ranger Single-cell software v6.0.0. The raw gene-barcode matrices were aligned to the Sscrofa11-1 genome.
Cell Ranger output was analyzed with the Seurat package v.4.0. using R v.4.0.2. Each individual data was filtered to keep cells with 200-7,000 genes, less than 99% of dropouts, and less than 5% of mitochondrial sequences.
Doublet cells were removed with DoubletFinder.
normalization and variance stabilization with SCTransform, and dimensionality reduction by Principal Component Analysis was performef for each sample separetely.
Global integration was done after normalization and identification of the 4000 most variable features of each dataset.
Genome_build: Sscrofa11-1
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
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| Submission date |
Feb 22, 2022 |
| Last update date |
Dec 31, 2022 |
| Contact name |
Kevin Lebrigand |
| Organization name |
IPMC/CNRS
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| Lab |
Functional Genomics Platform of Nice-Sophia-Antipolis, France.
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| Street address |
660 route des lucioles
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| City |
Valbonne - Sophia-Antipolis |
| ZIP/Postal code |
06560 |
| Country |
France |
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| Platform ID |
GPL20983 |
| Series (2) |
| GSE197187 |
Cystic fibrosis causes olfactory system defects by altering progenitor cell proliferation |
| GSE197189 |
Cystic fibrosis causes olfactory system defects by altering progenitor cell proliferation (scRNA-Seq) |
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| Relations |
| BioSample |
SAMN26174654 |
| SRA |
SRX14250256 |
| 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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