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| Status |
Public on Aug 14, 2019 |
| Title |
Sorted_cells_pool_1 |
| Sample type |
SRA |
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| Source name |
Enteroids
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| Organism |
Mus musculus |
| Characteristics |
tissue: Duodenum
mouse: female
age: 39 weeks
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| Growth protocol |
Mouse enteroids were generated as previously described (Sato et al., 2009) from small intestine (duodenum or jejunum) of CD1 Neurog3eYFP/+ mice (Mellitzer et al., 2004). The Matrigel domes containing enteroids were maintained in culture (37°C, 5% CO2) for 5-8 days in the following medium: basal medium (Advanced DMEM/F-12, HEPES 10mM (Gibco™), GlutaMax 2mM (Gibco™), penicillin/streptomycin 100U/mL (Gibco™)), added with B27 1X (Gibco™), N-acetyl-L-cysteine 1µM (Merck), human recombinant R-spondin-1 500ng/mL (PeproTech®), murine recombinant EGF 50ng/mL (PeproTech®), and human recombinant Noggin 100ng/mL (R&D Systems®).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
To dissociate the enteroids, the culture medium was removed and the Matrigel was broken. Then, the enteroids were digested to single cells in 500µL of TrypLE™ Express Enzyme (1X) (Gibco™) at 37°C for 20min. The dissociation was improved by passaging into a syringe mounted with a 18G needle. The digestion was stopped by addition of Advanced DMEM/F-12, fetal calf serum 10%, N-acetylcysteine 0,5mM, B27 1X. The cells were pelleted, washed 2 times and resuspended in the same medium. Finally, they are filtered on a 50 µm cell strainer. eYFP+ cells, from dissociated Neurog3eYFP/+ enteroids, were sorted directly into 96 well plates « Precise WTA Single Cell Encoding Plate » (BD™ Precise WTA Single Cell Kit, BD Genomics) (1 cell/well) using a FACS Aria Fusion (BD). DAPI was used to sort only living cells.
Cells were sorted directly into four 96 well plates « Precise WTA Single Cell Encoding Plate » (BDTM Precise WTA Single Cell Kit, BD Genomics) (1 cell/well) using a FACSAria II (BD). The library was prepared by the GenomEast Platform (IGBMC) following the protocol « Library Preparation with the BDTM Precise WTA Single Cell Kit User’s Guide » (910000014 Rev. 02 11/2016, BD Genomics).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Duodenum Enteroids ( mini-guts) from Ngn3eYFP mice (adult) were dissociated with TrypLE and mechanically. Single cells were filtered (50 µm) and sorted (FACS ARIA FUSION, noozle 100) directly in 96-well plates (BD, P/N 634100) and processed using in BD Precise WTA kit for single cells. 92 cells were collected, 4 wells left empty for controls.
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| Data processing |
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Fastq files were demultiplexed using the sample index encoded in reads 1.
Reads 2 having a bad quality (--quality-cutoff 20,20) or corresponding to polyA sequences or to adapters were removed using Cutadapt version 1.10.
Reads 2 mapping to rRNA were identified using Bowtie 2 version 2.2.8 and removed.
Reads 2 were aligned to mm10 genome using STAR version 2.5.3a.
Reads corresponding to technical duplicates were removed using UMI-Tools version 0.4.4. (method: "directional-adjacency")
Quantification of gene expression was performed using HTSeq version 0.6.1.post1 (parameter “--mode union”) and gene annotations from Ensembl release 90.
Cells were filtered in function of several quality criteria: % of reads uniquely aligned to genome ≥ 40% ; % of UMIs assigned to genes ≥ 50% ; Number of assigned UMIs per cell ≥ 200,000 ; % of UMIs assigned to mitochondrial genes < 20%.
Genome_build: mm10
Supplementary_files_format_and_content: The tab-delimited text file include raw UMI-counts for the 290 filtered cells.
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| Submission date |
Jun 19, 2019 |
| Last update date |
Aug 14, 2019 |
| Contact name |
Gérard Gradwohl |
| E-mail(s) |
gradwohl@igbmc.fr
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| Organization name |
IGBMC
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| Street address |
1 rue Laurent Fries
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| City |
Illkirch-Graffenstaden |
| State/province |
- |
| ZIP/Postal code |
67400 |
| Country |
France |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE132999 |
Intestinal endocrine subtype specification analyzed by Single Cell RNA-Seq in mouse enteroids/mini-guts. |
| GSE133038 |
Rfx6-regulated genes in the mouse intestine |
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| Relations |
| BioSample |
SAMN12095470 |
| SRA |
SRX6094327 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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