|
| Status |
Public on Dec 09, 2019 |
| Title |
Lgr5-EGFP-DTR, pylorus, Lgr5Hi, biological rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse adult pylorus
|
| Organism |
Mus musculus |
| Characteristics |
mouse model: Lgr5-EGFP-IRES-CreERT2
tissue: Pylorus
population: Lgr5Hi
|
| Treatment protocol |
Mice were not treated in any way, healthy mice were used for the experiment.
|
| Growth protocol |
Mice were bred and housed in Biological Resource Centre (Singapore). All animal experiments were approved by the Institutional Animal Care and Use Committee of Singapore. For all experiments adult animals (not selected for gender) with a minimum age of 7-8 weeks were used. Mouse models used were Lgr5-EGFP-IRES-CreERT (Barker 2007, Nature), Lgr5-EGFP-DTR (Tian 2011, Nature) and wildtype mice.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Murine pylorus was incubated in chelation buffer (5.6 mM sodium phosphate, 8 mM potassium phosphate, 96.2 mM sodium chloride, 1.6 mM potassium chloride, 43.4 mM sucrose, 54.9 mM D-sorbitol, 1 mM dithiothreitol) with 5 mM EDTA at 4oC for 2 hours. Glands were isolated by repeated pipetting of finely chopped pylorus tissue in cold chelation buffer. Chelation buffer containing isolated glands was filtered through 100μm filter mesh, and centrifuged at 720g at 4oC for 3 min. The pellet was resuspended in TrypLE (Life Technologies) with DNaseI (0.8U/μL)(Sigma) and incubated at 37oC for 10 min with intermittent trituration for digestion into single cells. Digestion was quenched by dilution with cold HBSS buffer. The suspension was centrifuged at 720g at 4oC for 3 min. For anti-Aqp5 antibody stain, the pellet was resuspended in HBSS with 2% fetal bovine serum (FBS, Hyclone) with anti-Aqp5-AF647 (Abcam, ab215225) at 1:500 dilution and incubated on ice at 30min in the dark. The pellet was subsequently washed twice with cold HBSS and spun at 800g for 3min at 4oC. The pellet was resuspended in HBSS with 2% fetal bovine serum (FBS, Hyclone). Before sorting, 1 μg/ml propidium iodide (Life Technologies) was added to the cell suspensions, filtered through a 40 μm strainer, and sorted on BD Influx Cell Sorter (BD Biosciences). Cells were collected in RLT Plus buffer (Qiagen) for RNA extraction or HBSS with 2% FBS and 1% PenStrep (Gibco) for organoid culture. Cells were lysed in RLT Plus buffer (Qiagen). RNA was subsequently isolated with RNeasy Universal Plus kit (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
RNA quality was first determined by assessing the integrity of the 28s and 18s ribosomal RNA bands on Agilent RNA 60000 Pico LabChips in an Agilent 2100 Bioanalyser (Agilent Technologies). A minimum of 2ng of RNA was used to generate SPIA-amplified cDNA using the Ovation Pico WTA system (Nugen Technologies). Five micrograms of SPIA-amplified purified cDNA was then fragmented and biotin-labelled with the Nugen Encore Biotin module (Nugen Technologies).
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| |
|
| Hybridization protocol |
Microarray was performed using the Affymetrix Mouse ST v2.0 GeneChips (Affymetrix), which consists of more than 28,000 probes for previously annotated genes. The individual microarrays were washed and stained in an Affymetrix Fluidics Station 450.
|
| Scan protocol |
Hybridized probe fluorescence was detected using the Affymetrix G3000 GeneArray Scanner. Image analysis was carried out on the Affymetrix GeneChip Command Console v2.0 using the MAS5 algorithm.
|
| Description |
Gene expression data from Lgr5Hi pyloric stem cells from Lgr5-EGFP-DTR mouse model
|
| Data processing |
CEL files were generated for each array and used for gene expression analysis. The CEL files were then processed in R (v3.2.3) with the Bioconductor (v3.2) libraries ‘oligo’ (v1.34.2), ‘pd.mogene.2.0.st’ (v3.14.1) and ‘limma’ (v3.26.8). We used Robust Multi-array Average (RMA)39 to perform background correction and normalization with the ‘rma’ function implemented in the ‘oligo’ package (‘target’ parameter was set to ‘core’ to obtain expression values at the gene level). The experimental design was stored as a single factor with individual levels for each combination of Lgr5-GFP level (high, low, negative) and treatment (tamoxifen and control). Linear models were fitted to the expression data with the function ‘lmFit’ (default parameters). The relevant contrasts were fitted with ‘contrasts.fit’ (default parameters); differential expression was tested with ‘eBayes’ (default parameters). Differential gene expression was analyzed using Partek Genomics Suite software (Partek).
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| |
|
| Submission date |
Oct 25, 2018 |
| Last update date |
Dec 09, 2019 |
| Contact name |
Simon Lieven Imanuel Johannes Denil |
| Organization name |
VUB/ULB
|
| Department |
University Hospital Brussels
|
| Lab |
BRIGHTcore
|
| Street address |
Laarbeeklaan 101
|
| City |
Brussels |
| ZIP/Postal code |
1090 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE121803 |
AQP5-Expressing Cells Serve as Stem Cells and Cancer Origins in the Mouse and Human Pyloric Stomach |
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