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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 16, 2024 |
| Title |
iPSC822_8m_transplant, snRNAseq |
| Sample type |
SRA |
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| Source name |
cortical organoid
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| Organism |
Homo sapiens |
| Characteristics |
tissue: cortical organoid
age: 8 months
cell line used to derive organoid: hESC
condition: transplant
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| Growth protocol |
Glia-enriched cortical organoids were generated using the reported protocol. Glia-enriched cortical organoids cultured in vitro were collected at 10 weeks and 5 months of differentiation for single nucleus RNA-seq. Glia-enriched cortical organoids were cultured in vitro for 8 to 10 weeks prior to implantation into the retrosplenial cortex of immunodeficient NSG mice. At 5, 6, and 8 months of differentiation, mice were sacrificed, and four organoid implants were dissected and pooled for single nucleus RNA-seq.
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| Extracted molecule |
total RNA |
| Extraction protocol |
In vitro organoids or fresh dissected in vivo brain organoids were pooled and lysed using a Dounce homogenizer in 1ml of freshly prepared cold lysis buffer (TrisHCl pH 7.4 10mM, NaCl 10mM, MgCl2 3mM, NP-40 0.1% in nucleus free water). Nuclei were centrifuged at 500g for 8 min and supernatant was discarded. Nuclei were then resuspended in freshly prepared cold nuclei wash & resuspension buffer (PBS, 1% BSA, RNase inhibitor 0.2U/μl) and filtered through a 35μm strainer (corning). Nuclei were centrifuged at 500g for 5 min and resuspended again in nuclei wash & resuspension buffer for a total of two washes. After the second wash, nuclei were resuspended in ice-cold nuclei wash & resuspension buffer at a concentration of 800-1,200 cells/μl, and approximately 17,400 cells per channel (to give estimated recovery of 10,000 cells per channel) were loaded onto a Chromium Next GEM Chip G (10x Genomics) and processed through the Chromium controller to generate single-cell gel beads in emulsion (GEMs).
Single nucleus RNA-seq libraries were prepared with the Chromium NextGEM Single Cell 3′ GEM Library & Gel Bead Kit v.3.1 (10x Genomics) as per manufacturer’s instruction.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Single-nucleus RNA-seq
10x Genomics
Human with minor mouse contamination
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| Data processing |
The Cell Ranger 6.0.1 pipeline (10x Genomics) was used to align reads from snRNA-seq collected from the transplants to the 10x hg19 human reference genome v1.2.0 and 10x mm10 mouse reference genome v1.2.0 and produce the associated cell-by-gene count matrix. Default parameters were used, except for –include-introns argument.
The Cell Ranger 6.0.1 pipeline (10x Genomics) was used to align reads from snRNA-seq collected from the in vitro organoids to the 10x hg19 human reference genome v1.2.0 and produce the associated cell-by-gene count matrix. Default parameters were used, except for –include-introns argument.
Unique molecular identifier (UMI) counts were analyzed using the Seurat R package v.4.0.1(Hao et al., 2021). Human nuclei (defined as nuclei with >95% genes mapped to hg19) were kept for subsequent analysis. Nuclei with <1% of mitochondrial contamination and between 500 and 6000 genes expressed were retained for further analysis.
The top 3000 variable genes for each line were then used to integrate sample datasets with the SCT method (SCTransform, SelectIntegrationFeatures, PrepSCTIntegration, FindIntegrationAnchors and IntegrateData functions). PCA was performed on the scaled integrated data for the variable genes and the top 30 principal components were used for the unsupervised clustering.
Cells were clustered in PCA space using the FindNeighbors function (top 30 principal components) and FindClusters function. UMAP plots were used to visualize variation in the data.
Assembly: hg19 and mm10
Supplementary files format and content: Tab-separated values files and matrix files (barcodes.tsv, features.tsv matrix.mtx).
Supplementary_files_format_and_content: integratedorganoid_withAnnot.txt: Contains annotations for 5-month-old organoids, as well as 5-month-old, 6-month-old, and 8-month-old transplants.
Supplementary_files_format_and_content: 10wOrg_withAnnot.txt: Contains annotations for 10-week-old organoids.
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| Submission date |
Oct 25, 2022 |
| Last update date |
Sep 13, 2024 |
| Contact name |
April Elizabeth Williams |
| E-mail(s) |
apriljack06@gmail.com, awilliams@salk.edu
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| Phone |
7345461645
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| Organization name |
Salk Institute for Biological Studies
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| Department |
IGC
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| Street address |
10010 N Torrey Pines Rd
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| City |
San Diego |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE185472 |
Morphological diversification and functional maturation of human astrocytes in glia-enriched cortical organoids transplanted in the mouse brain |
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| Relations |
| BioSample |
SAMN31445429 |
| SRA |
SRX18025331 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6679387_822_8m_GOGNT_barcodes.tsv.gz |
38.8 Kb |
(ftp)(http) |
TSV |
| GSM6679387_822_8m_GOGNT_features.tsv.gz |
518.6 Kb |
(ftp)(http) |
TSV |
| GSM6679387_822_8m_GOGNT_matrix.mtx.gz |
58.9 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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