Gene Expression
Nebulas
Gene Expression NebulasSummary: The Recombination Activation Gene, RAG1, expression of which presages T-cell receptor gene rearrangement, is a key marker of T-cell commitment. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cells genesis in the context of haemtopoietic organoids. We show that T-cell commitment occurs concomitantly with the emergence of blood cells from AGM-like haemogenic endothelium, predating the surface expression of CD5 and CD7. In this system, RAG1 marks an early haematopoietic progenitor emerging from SOX17+ endothelium, prior to down regulation of CD90 and VCAM and upregulation of the blood cell marker, CD45. Sort and re-culture experiments show that early RAG1+ cells possess T-cell, B-cell, myeloid and erythroid potential. However, under conditions that favor T-cell development, early RAG1+ cells progress to the CD4+CD8+CD3+ stage, vindicating their classification as bone fide T-cell progenitors. These observations suggest that like the zebrafish and mouse, humans can execute a non-HSC derived wave of T-cell development that includes a multipotent RAG1+ progenitor.
Overall Design: sinlge cell transcriptional profiling of RAG1+ cells generated early on (day 20 and 16) during air-liquid interface culture of haemopoietic organoids
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| Growth Protocol: | Induced pluripotent stem cell lines were differentiated as embryoid bodies (EBs) for 8 days in presence of appropriate supplements. The EBs were then transferred onto Corning transmembranes to grow in air liquid interface system until the end of experiments. |
| Treatment Protocol: | - |
| Extract Protocol: | iPSC derived RAG1:GFP+ purified cells were submitted to the Australian Genome Research Facility (AGRF) for single cell RNA sequencing using 10x Genomics Chromium system. |
| Library Construction Protocol: | The sequencing and data generation was performed using AGRF- Illumina bcl2fastq pipeline version 2.20.0.422, via generation of 100bp Paired End reads and sequencing through Illumina HiSeq platform. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Specific |
| 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 | Condition 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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