characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2021-11-03T00:30:05Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2021-11-03T00:30:05Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA10167113
characteristics.characteristic: External Id
characteristics.text: ETH Zurich, D-BSSE
characteristics.characteristic: INSDC center name
characteristics.text: 2021-11-03T00:30:05Z
characteristics.characteristic: INSDC first public
characteristics.text: 2021-11-03T00:30:05Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS7823588
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-10972:SLICE2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: 409B2-iCas9
characteristics.characteristic: cell line
characteristics.text: brain cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000129
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Organoids were embedded in pre-chilled Optimal Cutting Temperature (OCT). The sample was then set into a dry ice bath with isopentane until frozen and stored at -80°C. Cryosections were cut at 10um thickness, adhered to ST slides (10x) and stored at -80°C until the following day. Tissue slices were fixed in cold methanol, before being stained in Hematoxylin and eosin. ST slides were imaged as recommended on a Nikon T2i at 20x using a tile scan over all slice sections. 2000 iTracer+ iCRISPR iPSCs per well in a 96-well plate were seeded and differentiated into cerebral organoids using a whole organoid differentiation protocol by Lancaster et al. (Nature Protocols, 2014). Organoids were scarred by activation of inducible Cas9 at day 15. Scarring was achieved by first selecting the organoid to be scarred and transferring it to a 6-24-well plate (depending on organoid size) filled with 8ug doxycycline scarring media. Organoids were incubated in scarring media for 24 hours before returning to base media without doxycycline. Tissue slices were permeabilized to obtain RNA, optimal permeabilization conditions were determined by using the Tissue Optimization Kit (10x) and was found to be 24 minutes. Spot-captured RNA was reverse transcribed before second strand synthesis and cDNA denaturation. qPCR was used to determine the optimal number of cDNA amplification cycles as recommended by the manufacturer. cDNA was amplified using 17-18 cycles. Libraries were constructed following the 10X Visium library construction protocol v1. Barcode and scar regions were further amplified from 60-70ng of cDNA. Afterwards, a PCR was applied to introduce library specific sequencing primers as provided by 10X. Libraries were quantified and checked for size distribution using Agilent Bioanalyzer DNA High Sensitivity Assays.
characteristics.characteristic: description
characteristics.text: normal
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000461
characteristics.characteristic: disease
characteristics.text: iTracer
characteristics.characteristic: genotype
characteristics.text: cerebral organoid
characteristics.characteristic: growth condition
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: induced pluripotent stem cell
characteristics.characteristic: progenitor cell type
characteristics.text: SLICE2
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0008205
characteristics.characteristic: sampling site
characteristics.text: 60
characteristics.unit: day
characteristics.characteristic: sampling time point
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: SLICE2
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0008205
characteristics.characteristic: title