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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 29, 2020 |
| Title |
organoid, RSPO1 |
| Sample type |
SRA |
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| Source name |
Intestinal adenoma organoid culture
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: ApcMin/+
tissue: intestinal adenoma
age: 17 weeks
treatment: RSPO1-Fc fusion protein
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| Treatment protocol |
Organoids were cultured with or without RSPO1-Fc fusion protein (10 ug/ml)
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| Extracted molecule |
total RNA |
| Extraction protocol |
In order to obtain single cell solution, itestinal organoids derived from ApcMin/+ mice treated with or without RSPO1-Fc fusion protein were incubated in Hanks' balanced salt solution (HBSS, 14175-053, Gibco) containing 1 mg/mL collagenase type I (LS004196, Worthington), 1 mg/mL collagenase H (11074032001, Roche), 4 mg/mL dispase II (04942078001, Sigma) and 1000 U/mL benzonase (sc-202391, ChemCruz) for 30 min at 37°C with gentle shaking, followed by 10 min incubation with trypsin. Cells were transferred into 0.04% BSA-HBSS for analysis.
Single cells were analyzed using the 10x Chromium Single-Cell 3′RNA-sequencing system (10x Genomics, Pleasanton, CA, USA) with the Reagent Kit v2 as instructed by the manufacturer. Briefly, the cells were first loaded into Chromium Single-Cell Chip v2 (10x Genomics, Pleasanton, CA, USA), followed by generation of Gel beads in EMulsion (GEM) aiming at 5000 cell captures per sample. This was followed by cDNA purification, amplification (12 cycles), and library construction (sample index PCR 14 cycles) as instructed by the manufacturer.
The sample libraries were sequenced on an Illumina NovaSeq 6000 sequencer using read lengths of 26bp (Read 1), 8bp (i7 Index), 0 bp (i5 Index) and 91bp (Read 2), resulting in an average of following reads per cell: 58 414 (Ctrl), and 131 291 (RSPO1). Chromium single-cell 3’ RNA-sequencing barcodes and read data were demultiplexed and converted to FASTQ files, and aligned reads and gene-cell matrices were generated using Cell Ranger v 2.1.1 mkfastq and count pipelines (10x Genomics, Pleasanton, CA, USA). Reads were aligned to mouse reference genome mm10.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
ApcMin/+ mouse intestinal adenoma organoids cultured with 10 mg/ml RSPO1-Fc fusion protein
MinOrganoidsCombi.Rdata
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| Data processing |
The Cell Ranger v 2.1.1 mkfastq and count pipelines (10x Genomics, Pleasanton, CA) were used to demultiplex and convert Chromium single-cell 3’ RNA-sequencing barcodes and read data to FASTQ files and to generate aligned reads and gene-cell matrices.
Reads were aligned to mouse reference genome mm10
We used the Seurat R package 3.1.1 for quality control, filtering, and analysis of the data
Cells were filtered based on unique feature counts and the percentage of mitochondrial genes
Cells with more than 15% of mitochondrial genes were filtered out
The expression matrix was further filtered by removing genes with expression in less than five cells and cells with less than 500 distinct expressed genes.
Filtered data was combined for CCA and further analysis, and saved as MinOrganoidsCombi.Robj (supplementary file has .Rdata extension)
Genome_build: mm10
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| Submission date |
Feb 28, 2020 |
| Last update date |
Feb 29, 2020 |
| Contact name |
Marianne Lähde |
| E-mail(s) |
[email protected]
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| Organization name |
University of Helsinki
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| Department |
Faculty of Medicine, Translational cancer medicine program
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| Lab |
Alitalo
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| Street address |
Haartmaninkatu 8
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| City |
Helsinki |
| ZIP/Postal code |
00290 |
| Country |
Finland |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE146099 |
Single cell changes in RSPO1-Fc treated intestinal adenoma organoids from ApcMin/+ mice |
| GSE146139 |
RSPO1 suppresses growth of mouse intestinal adenomas |
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| Relations |
| BioSample |
SAMN14245972 |
| SRA |
SRX7817733 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4365351_Rspo_barcodes.tsv.gz |
2.2 Mb |
(ftp)(http) |
TSV |
| GSM4365351_Rspo_genes.tsv.gz |
212.7 Kb |
(ftp)(http) |
TSV |
| GSM4365351_Rspo_matrix.mtx.gz |
87.8 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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