Gene Expression
Nebulas
Gene Expression NebulasSummary: The scRNA-seq analysis of 75 day old human cerebral organoids grown at the air-liquid interface (ALI-COs) reveals six well-defined major clusters. Cell-type and maturity markers define a (1) distinct population of deep layer subcortical projection neurons, (2) upper layer intracortical (callosal) projection neurons and intermediate progenitors, (3) ventricular and subventricular zone radial glial cells, (4) more mature upper and deep layer neurons, (5) interneurons and (6) actively dividing cells with intermediate progenitor and radial glia markers. In addition, the gene expression profile of these clusters corresponds with their expected functional characteristics appropriate to their maturity. Altogether, our data reflect a full repertoire of cortical neuronal and progenitor identities corresponding with the stages of cortical development in age-matched fetal brains.
Overall Design: Single cell RNA-sequencing data was generated using the 10X Genomics Chromium Single Cell 3' Chip and workflow.
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| Growth Protocol: | H1 and H9 human embryonic stem cell-derived cerebral organoids were generated and grown as slice cultures as described in Giandomenico et al., 2018. Organoids were supplemented with serum-free slice culture medium (SFSCM): Neurobasal (Invitrogen, cat. #21103049), 1:50 (v/v) B27+A (Invitrogen, cat. #17504044), 0.5% (w/v) glucose, 1X (v/v) Glutamax supplemented with Antibiotic-Antimycotic (ThermoFisher, cat. #15240062). The cultures were maintained in SFSCM at 37 ℃ and 5% CO2 with daily media changes. |
| Treatment Protocol: | - |
| Extract Protocol: | The cell suspension (34μl with a total of 7000 cells) was loaded onto a 10X Genomics Chromium Single Cell 3' Chip with the appropriate amount of Mastermix. RNA extraction was performed following the same 10X Genomics Chromium workflow. |
| Library Construction Protocol: | The reverse transcriptase reaction and subsequent amplification was carried out in a C1000 Touch Thermal Cycler (Biorad), with the cDNA being amplified for 12 cycles. Before sequencing, libraries were quality tested using the 2100 Bioanalyzer Instrument (Agilent) and their concentration was measured by qPCR. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 4000 |
| 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 | 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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