characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-10-14T00:44:16Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-10-14T00:44:16Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA110694750
characteristics: External Id
characteristics: UoH
characteristics: INSDC center name
characteristics: 2022-10-14T00:44:16Z
characteristics: INSDC first public
characteristics: 2022-10-14T00:44:16Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS12792301
characteristics: SRA accession
characteristics: E-MTAB-12122:Sample 11
characteristics: Submitter Id
characteristics: < 24
characteristics: month
characteristics: age
characteristics: ArrayExpress
characteristics: broker name
characteristics: intestinal crypt stem cell of small intestine
characteristics: cell_type
characteristics: house mouse
characteristics: common name
characteristics: Protocols: Mouse small intestines were flushed with cold PBS, opened and mucus was removed. Intestine was cut to small fragments and incubated with several changes of 10 mM EDTA in PBS on ice for 2 h. Epithelium was detached by vigorous shaking. To enrich crypts, tissue suspension was filtered through 70 μm nylon mesh. Enriched crypts were washed once with cold PBS and plated into 60% Matrigel (BD Biosciences) with ENR media. 10 μM Y-27632 was added to the media for the first 2 days. To isolate single cells, isolated crypts were dissociated in TrypLE Express (Gibco) with 1000 U/ml of DNaseI (Roche) +32 °C for 90 seconds. Cells were washed and stained with antibodies anti-CD31-PE (Biolegend, Mec13.3), anti-CD45-PE (eBioscience, 30-F11), anti-Ter119-PE (Biolegend, Ter119), anti-EpCAM-APC (eBioscience, G8.8) and anti-CD24-Pacific Blue (Biolegend, M1/69). Finally, cells were resuspended to SMEM media (Sigma) supplemented with 7-AAD (Life) for live cell separation. Cells were sorted by using FACSAria II or FACSAria Fusion (BD Biosciences). Total RNA from sorted cells were isolated by Trizol purification. Samples were first treated with HL-dsDNAse (ArticzymesP/N 80200-050) to remove residual DNA. An Ovation Universal RNA-Seq System kit was used for Illumina library preparations (NuGEN Technologies Inc., CA, USA). Purified total RNA (100 ng) was used and primers for ribosomal removal were designed and used as outlined in the kit manual. Libraries were purified with AMPure XP beads (Beckman Coulter Inc., MA, USA),
characteristics: description
characteristics: old
characteristics: developmental stage
characteristics: Lgr5-EGFP-IRES-creERT2
characteristics: genotype
characteristics: NP275
characteristics: individual
characteristics: Mus musculus
characteristics: organism
characteristics: Mus musculus
characteristics: organism
characteristics: epithelium of small intestine
characteristics: organism part
characteristics: Mus musculus
characteristics: scientific_name
characteristics: female
characteristics: sex
characteristics: C57BL/6J
characteristics: strain
characteristics: Sample 11
characteristics: title