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| Status |
Public on Jan 12, 2023 |
| Title |
IBD-drop-d7, replicate 1, scRNAseq |
| Sample type |
SRA |
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| Source name |
terminal ileum
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| Organism |
Homo sapiens |
| Characteristics |
tissue: terminal ileum
cell type: intestinal epithelial cells
treatment: inflammatory bowel disease (IBD)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Enteroid lines are generated from terminal ileum mucosal biopsies. Briefly, two biopsy tissue fragments were rinsed 3 times in 1 ml cold sterile PBS, then incubated in cold chelation buffer for 30 minutes on a turntable in a cold room, followed by mechanical dis-sociation (scraping) of epithelial layer. The fragments were strained through a 100 μm strainer to deplete the villi and resuspended in 80% Matrigel, then seeded at the density of 50-200 crypts per 30 μl drop. The droplets solidified at 37ºC for 30 minutes, and 500 μl human IntestiCult (STEMCELL Technologies; complete when supplemented with Penicillin-Streptomycin (Gibco)) was added per well. Y-27632 (SelleckChem; final conc. 10 μM) was added to culture medium at seeding only.
Single-cell suspension for each organoid sample was loaded onto a separate channel of a Chromium 10X Genomics Single Cell 3’ Reagent Kit v2 library chip (10X Genomics) according to the manufacturer’s protocol. RNA transcripts from single cells were uniquely barcoded and reverse-transcribed. cDNA sequencing libraries were prepared according to the manufacturer’s protocol (10X user guide for library prep) and sequenced on an Illumina NovaSeq 6000 using an S1 100 cycles flow cell v1.5. Library quality control was done using Agilent TapeStation for sizing (bp) and KAPA qPCR for concentration (nM).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10X Genomics
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| Data processing |
Raw sequence reads data were processed using the CellRanger pipeline (10X Genomics, v.5.0.0) for demultiplexing and aligned to the human genome GRCh38 transcriptome. Sample data was aggregated using the CellRanger aggr pipeline and libraries were normalized for sequencing depth across the sample set. A total of 5 organoid sample count matrices were merged together for cell type identification and direct comparisons. For initial filtering, cells with a smaller number (< 200) of uniquely detected genes were excluded. Then, cells with a proportion of unique molecular identifiers (UMIs) count attributable to mitochondrial genes (> 25%) were excluded from the analysis. Finally, highly variable genes were used for dimension reduction, and the results were visualized via UMAP.
Assembly: human genome GRCh38
Supplementary files format and content: barcodes, features, and matrix files
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| Submission date |
May 19, 2022 |
| Last update date |
Jan 12, 2023 |
| Contact name |
Dan Dongeun Huh |
| Organization name |
University of Pennsylvania
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| Street address |
210 South 33rd Street 315S Skirkanich Hall
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| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE203380 |
Geometric engineering of organoid culture for enhanced organogenesis in a dish |
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| Relations |
| BioSample |
SAMN28548424 |
| SRA |
SRX15372279 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6170832_S1_barcodes.tsv.gz |
18.5 Mb |
(ftp)(http) |
TSV |
| GSM6170832_S1_features.tsv.gz |
287.6 Kb |
(ftp)(http) |
TSV |
| GSM6170832_S1_matrix.mtx.gz |
139.7 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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