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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 10, 2019 |
| Title |
E8.75 Gut tube anterior - mid gut Rep 1 |
| Sample type |
SRA |
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| Source name |
E8.75 Gut tube anterior - mid gut Rep 1
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| Organism |
Mus musculus |
| Characteristics |
developmental stage: E8.75
tissue: Anterior - Mid gut tube
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| Growth protocol |
Mice were maintained under a 12 hr light-dark cycle and checked for vaginal plugs in the morning. All embryos for this study were obtained by natural matings of virgins 6-8 weeks of age. Embryos at noon on the day of detecting copulation plugs were estimated at E0.5. Uteri were flushed with flushing and holding medium (FHM, Millipore) as previously described. Embryos were subsequently washed in 2-3 drops of FHM and kept in drops of FHM covered with mineral oil (Sigma) on ice prior to single-cell dissociation. Post-implantation embryos were dissected in DMEM/F12 (Life Technologies)/5% Newborn calf serum (Life Technologies) and staged according to Downs and Davies 6 or by somite number.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
13ss (E8.75) embryos were used as the terminal end point of this study since this is the latest time one can confidently assign cells in the gut tube with a VE-vs.-DE identity using either the Afp-GFP reporter or the Ttr-Cre driver crossed to a Cre reporter, since by 18-20ss (E9.0) both Afp and Ttr genes, and cis-regulatory elements used in the respective transgenic lines of mice, become activated in gut endoderm derivatives including the emergent liver. To obtain single cells from 13ss gut tubes, Afp-GFPTG/+ embryos were dissected out of the uterus, and extra- embryonic membranes and heads were removed. Torsos washed in three drops of DMEM/F12 on ice and subjected to Pancreatin/Trypsin treatment (2.5% Pancreatin / 0.5% Trypsin in PBS) for 5 min (exact time was batch dependent and empirically tested) on ice and then washed in three drops of DMEM/F12, 10% Newborn calf serum on ice. Gut tubes were dissected out using Tungsten needles (FST Cat No. 10130-10) and washed in cold DMEM/F12. Gut tubes were then incubated for 20 min at 37°C in Accutase/0.25% Trypsin (1:2) for the dissociation of single cells. To obtain single cells from definitive and visceral endoderm cell from stages E7.5 and visceral endoderm cells from E6.5, embryos were washed in three drops of DMEM/F12 on ice and subjected to Pancreatin/Trypsin treatment (2.5% Pancreatin / 0.25% Trypsin in PBS) for 3 min (E7.5) and 45s (E6.5) on ice and then washed in three drops of DMEM/F12, 10% Newborn calf serum on ice. The endoderm layer was removed from the rest of the tissue using Tungsten needles and subsequently washed in cold DMEM/F12 and incubated for 20 min at 37°C in Accutase/0.25% Trypsin (1:2). For E5.5 (defined as the stage when the DVE/AVE was distally positioned, observed as a thickening of the emVE epithelium), whole embryos were collected, and Reichert’s membrane removed with Tungsten needles. Embryos were washed in cold DMEM/F12 and then incubated in 0.25% Trypsin for 5 min at 37°C. To dissociate the tissue into a single cell suspension, DMEM/F12, 20% Newborn calf serum, 4mM EDTA were added in 1:1 ratio. Cell clumps were dissociated into single cells using a mouth pipet and 75mm glass capillaries. Subsequently, the single cell suspension was filtered through a FlowMI cell strainer (40μm) to remove debris. Single cells were spun down at 450g for 4 min at room temperature and then cell numbers were determined using a Neubauer hemocytometer. For pre-implantation embryo dissociations, embryos were incubated in 0.5% Trypsin-EDTA (Invitrogen) at 37°C for 3 minutes before transferring to PBS supplemented with 0.5mM EDTA (Invitrogen) and 4% BSA (Sigma) for mechanical dissociations. Trypsin-treated embryos were dissociated by repeated pipetting (trituration) using pulled capillaries (Sutter Instruments) attached to a mouth pipet.
RNA concentration and quality were assessed, and cDNA libraries construction and sequencing were done by the Genomics and Epigenomics Core Facility at Weill Medical College, Cornell University, New York
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Bulk RNA-seq data was aligned to the mm38 mouse genome using STAR and reads that mapped to multiple genomic locations were filtered out.
Gene expression counts for each sample were determined using the summarizeOverlaps function of the GenomicRanges package using Ensembl annotations.
Genome_build: mm38
Supplementary_files_format_and_content: Vector of unnormalized gene expression counts in gzipped csv format
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| Submission date |
Nov 29, 2018 |
| Last update date |
Apr 12, 2019 |
| Contact name |
Manu Setty |
| E-mail(s) |
[email protected]
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| Organization name |
Fred Hutchinson Cancer Center
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| Department |
Basic Sciences Division
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| Street address |
1100 Fairview Ave N
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE123124 |
The emergent landscape of the mouse gut endoderm at single-cell resolution |
| GSE123135 |
The emergent landscape of the mouse gut endoderm at single-cell resolution |
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| Relations |
| BioSample |
SAMN10498847 |
| SRA |
SRX5078882 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3496085_E8.75_anterior_midgut_rep1.csv.gz |
135.8 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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