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Sample GSM1900658

Query DataSets for GSM1900658

Status

Public on May 11, 2016

Title

Trop2+ cells E14.5 Small intestine Sample1

Sample type

SRA

Source name

Trop2+ small intestine

Organism

Mus musculus

Characteristics

strain background: mixed
developemental stage/age: E14.5 embryo
tissue: small intestine
cell type: fetal Trop2 epithelial intestinal
passages: tissue-sorted cells

Treatment protocol

Adult Lgr5-DTR heterozygous mice were injected with diphetheria toxin (a dose of 50 µg/kg of body weight) at day 1, 2 and 4. Stomach were harvested at day5 for FACS sorting.

Growth protocol

Ex vivo cultures: Embryonic stomach and intestine tissue were dissociated and epithelial samples were cultured according to the protocol reported by Sato et al (2010). Specifically, the culture medium used for growing gastric spheroids consisted in Advanced-DMEM/F12 medium supplemented with 2 mM L-Glutamine, N2 and B27 w/o vit.A (Invitrogen), gentamycin, penicillin-streptomycin cocktail, 10 mM HEPES, and 1 mM N acetyl cysteine. Growth factors were added at a final concentration of : 50 ng/ml EGF and 100 ng/ml Noggin (both from Peprotech), and 100 ng/ml CHO-derived R-spondin1 (R&D System). Culture medium was changed each other day and after 5-6 days in culture, spheroids were harvested, mechanically dissociated and replated in fresh Matrigel (BD). Isolation of Trop2+ve and Lgr5-GFP+ve cells from fetal stomach and adult pyloric stomach from Lgr5-DTR, after Lgr5 cells ablation, was performed by FACS. Proximal and distal areas of E14.5 embryos stomach (pool of 12, 18 and 19 embryos from given litter per experiment, n=3 independent experiments), were completely dissociated with the Stem Pro accutase cell dissociation reagent (Thermo electron) at 37°C and passed through a 40-μm nylon cell strainer (Greiner). For adults Lgr5-DTR, antral glands were isolated as previously described by Barker et al. (Barker et al., 2010). Glands were after incubated in Stem Pro accutase cell dissociation reagent until total dissociation and passed through a 40-μm nylon cell strainer. Cells from both types of isolation procedures were incubated with fluorochrome-conjugated anti-Trop2-APC antibody, or the relevant isotype control (R&D systems) in PBS-2% BSA-2mM EDTA for 45 min on ice and sorted by using the Facs Aria I (BD Biosciences). Lgr5-GFP+ve cells were directly sorted using the FITC channel. Sorted cells were directly collected for RNA analysis over Qiazol lysis reagent (Qiagen).

Extracted molecule

total RNA

Extraction protocol

900 ng of total RNA extracted with the miRNA isolation kit (Ambion, Life Technologies), were checked for quality by Bioanalyzer (Agilent).
TruSeq® Stranded mRNA libraries were prepared for sequencing using standard Illumina protocols.
paired-end libraries 2x 100 bases or 2x 125 bases

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1500

Description

E14.5 embryo

Data processing

Approximately 25 million of paired-end reads per sample were mapped against the mouse reference genome (GRCm38.p4/mm10) using STAR software to generate read alignments for each sample.
Annotations Mus_musculus.GRCm38.79.gtf were obtained from ftp.Ensembl.org.
After transcripts assembling, gene level counts were obtained using HTSeq.
Genome_build: GRCm38.p4/mm10
Supplementary_files_format_and_content: tab-delimited text files include CPM (count per million) values for each Sample.

Submission date

Oct 02, 2015

Last update date

May 15, 2019

Contact name

Frederick Libert

E-mail(s)

flibert@ulb.ac.be

Organization name

ULB

Department

IRIBHM