characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-02-21T12:19:11Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-02-21T12:19:11Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112294297
characteristics.characteristic: External Id
characteristics.text: University College London, Institute of Ophthalmology
characteristics.characteristic: INSDC center name
characteristics.text: 2023-02-21T12:19:11Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-02-21T12:19:11Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14405127
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12467:Day30_MG2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: RC9
characteristics.characteristic: cell_line
characteristics.text: Mueller cell
characteristics.characteristic: cell_type
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: CD29+/CD44+ cells were isolated from retinal organoids derived from human embryonic stem cells. Retinal organoids at different stages of development, including days 10, 20, 30, 50, 70 and 90 after initiation of retinal differentiation, were dissociated into single cells using a gentle cell dissociation reagent (Stem Cell Technologies, UK) (5mins at 37°C), and plated onto fibronectin coated tissue culture plates (50µg/ml) in the presence of fibroblast growth factor (FGF) (20ng/ml) and epidermal growth factor (EGF) (20ng/ml) in DMEM. After 3 days in culture, cells expressing the surface markers CD29 and CD44 that had selectively adhered to fibronectin, were detached, washed and total RNA was extracted. Retinal organoids were cultured according to the following publication DOI: 10.1002/sctm.18-0263 Total RNA was extracted using Qiagen Rneasy micro plus kit (Cat. 74034, Qiagen, UK) Samples were processed using the KAPA mRNA HyperPrep Kit (p/n KK8580).purified mRNA was fragmented using chemical hydrolysis (heat and divalent metal cation) and primed with random hexamers. Strand-specific first strand cDNA was generated using Reverse Transcriptase in the presence of Actinomycin D.The second cDNA strand was synthesised using dUTP in place of dTTP, to mark the second strand. The resultant cDNA was then “A-tailed” at the 3’ end to prevent self-ligation and adapter dimerisation. Full length xGen adaptors (IDT), containing two unique 8bp sample specific indexes, a unique molecular identifier (N8) and a T overhang were ligated to the A-Tailed cDNA. Successfully ligated cDNA molecules were then enriched with limited cycle PCR (12 cycles). The high fidelity polymerase employed in the PCR is unable to extend through uracil. This means only the first strand cDNA is amplified for sequencing, making the library strand specific (first-strand).
characteristics.characteristic: description
characteristics.text: embryo
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: retinal organoid
characteristics.characteristic: growth condition
characteristics.text: CD29 positive; CD44 positive
characteristics.characteristic: immunophenotype
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: inner cell mass
characteristics.characteristic: organism part
characteristics.text: embryonic stem cell
characteristics.characteristic: progenitor cell type
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: Day30_MG2
characteristics.characteristic: title