characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-02-24T00:19:09Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-02-24T00:19:09Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112647750
characteristics.characteristic: External Id
characteristics.text: D-BSSE, ETH ZURICH
characteristics.characteristic: INSDC center name
characteristics.text: 2023-02-24T00:19:09Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-02-24T00:19:09Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14645473
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12622:RO_CROP_Exp1_guide
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: 409b2-iCas9
characteristics.characteristic: cell_line
characteristics.text: retinal cell
characteristics.characteristic: cell_type
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Retinal organoids were dissociated with the papain-based dissociation kit (Miltenyi Biotec, 130-092-628) described previously, and GFP+ cells were enriched by fluorescence activated cell sorting (FACS) The retinal organoids were generated with 1500 iPS cells per micro-well and treated with BMP4 (55ng/ml, Biotechne #314-BP) on day 6 with half medium change on day 9, 12 and 15. Retinal organoids derived from 409B2-iCas9 line at 19 weeks of development were infected with the lentiviral pool described above. 20 organoids were individually infected in ultra-low attachment 96 well plates (Costar). Each organoid was infected with 1ul of LV (titer = 5.61E+08 TU/ml) in 100ul medium. After 4hrs, 150 ul of medium were added for O/N incubation. The day after, organoids were moved to 10cm plates and treated with doxycycline 4ug/ml for 1 week to induce Cas9. After three weeks, 10 organoids were dissociated with the papain-based dissociation kit (Miltenyi Biotec, 130-092-628) described previously, and GFP+ cells were enriched by fluorescence activated cell sorting (FACS), (RO_CROP_Exp1). The other 10 organoids were processed five weeks after infection (RO_CROP_Exp2). Single cell RNA-seq libraries were generated with the Chromium Single Cell 3’ V3.1 Library & Gel Bead Kit. Single cell RNA-seq libraries were generated with the Chromium Single Cell 3’ V3.1 Library & Gel Bead Kit. In addition, guide RNA sequences from single cells were amplified from 30ng of scRNA-seq cDNA as described before. In brief, The first PCR amplifies a broad region targeting the outer part of the U6 promoter. The second, nested, PCR targets the inner portion of the U6 promoter adjacent to the guide sequence and adds a TruSeq Illumina i5 adapter. Finally, a third PCR adds Illumina sequencing i7 adaptors. PCRs were monitored by qPCR to avoid over-amplification. The samples were purified using SPRI beads (Beckman Coulter).
characteristics.characteristic: description
characteristics.text: embryo
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: mixed lentiviral pool against OTX2, NRL, CRX, VSX2, and PAX6 with guideRNA
characteristics.characteristic: genotype
characteristics.text: retinal organoid
characteristics.characteristic: growth condition
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: skin
characteristics.characteristic: organism part
characteristics.text: induced pluripotent stem cell
characteristics.characteristic: progenitor cell type
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: RO_CROP_Exp1_guide
characteristics.characteristic: title