Gene Expression
Nebulas
Gene Expression NebulasSummary: The development of digestion tract is critical for proper digestion of food and absorbance of nutrient for an individual. It includes the spatial segregation into esophagus, stomach, small and large intestine. The temporal-spatial gene expression profiles of human digestion tract in vivo have never been analyzed at single-cell resolution.Here we analyzed esophagus, stomach, small intestine (SI) and large intestine (LI) from multiple human embryos between 6 and 25 weeks of gestation by single cell RNA-seq analyses.We firstly identified 51 clusters of different types of cells using t- Distributed Stochastic Neighbor Embedding (t-SNE) analysis.Moreover,cell type of each organ were identified according to the known marker genes and new cell types were further analyzed.The dynamic change of each cell type were tracked during the human embryonic digestive tract development.We found HOX family geens paly different roles in the regulation of digestion tract developemnt. In addition, Hedgehog,TGFå°¾ and BMP signaling pathway are essential for SI and LI development in human fetal. The function of nutrient digestion and absorption was increased as the SI development in human fetal.Finally, we demonstrated that the immune system was established at late stage of human fetal and verified the immune-like cells,such as T cell, B cells and macrophage. In summary, by using single cell RNA seq technique, we identified the waves of signaling pathways and critical cell types for the human digestion tract development.
Overall Design: Single cell transcriptome profiles of four main human organs (esophagus, stomach, small and large intestine) from 6 week to 25 week gestation of digestion system were generated by next generation sequencing using Illumina HiSeq X Ten
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| Growth Protocol: | For every embryo, the main organs of digest system, including esophagus, stomach, small intestine (SI), large intestine (LI), were dissected. For 11W to 25W small intestine, the villi and crypt were separated by shaving off the villi carefully with a surgical blade. The mesenterium of small intestine and large intestine were torn off carefully under a microscope. The middle section of dissected esophagus, SI and LI was used to collect single cells. And the corpus of stomach was used to collect single cells. The selected tissue pieces were washed by PBS for several times considering there many microorganisms and mucus in digest system even in the early development stage. Then the selected tissue pieces were minced to small pieces by surgical scissors, after which the minced tissues were digested to single cells by using 2mg/mL Collagenase/Dispase for 30-40 min at 37. When digest intestine crypt, 2mM EDTA was firstly used to insure the release of the cells from intestine crypt. 40m strainer was used to filter large tissue debris. Then the cell suspension in the solution of Collagenase/Dispase was centrifuged at 800g for 5min at 4 and the cell pellet was re-suspended in 1% HSA. |
| Treatment Protocol: | - |
| Extract Protocol: | After pick single cells using mouth pipette, they were transferred to cold scRNA-seq lysis buffer immediately. Subsequently, the lysis buffer were shocked for 3min at 72 to make sure the release of RNA. |
| Library Construction Protocol: | - |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | SINGLE |
| Library Strand: | Reverse; - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 4000 |
| Strand-Specific: | Specific; Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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