title: Day50_MG1
organism: Homo sapiens
description: 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-02-21
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-02-21
attributes.tag: External Id
attributes.value: SAMEA112294299
attributes.tag: INSDC center alias
attributes.value: University College London, Institute of Ophthalmology
attributes.tag: INSDC center name
attributes.value: University College London, Institute of Ophthalmology
attributes.tag: INSDC first public
attributes.value: 2023-02-21T12:19:11Z
attributes.tag: INSDC last update
attributes.value: 2023-02-21T12:19:11Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12467:Day50_MG1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: RC9
attributes.tag: cell_type
attributes.value: Mueller cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: retinal organoid
attributes.tag: immunophenotype
attributes.value: CD29 positive; CD44 positive
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: progenitor cell type
attributes.value: embryonic stem cell
attributes.tag: sample name
attributes.value: E-MTAB-12467:Day50_MG1
attributes.tag: scientific_name
attributes.value: Homo sapiens