characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-08-18T00:13:25Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-08-25T00:13:43Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110471146
characteristics.characteristic: External Id
characteristics.text: ETH Zurich, DBSSE
characteristics.characteristic: INSDC center name
characteristics.text: 2022-08-18T00:13:25Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-08-25T00:13:43Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12569172
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12002:MULTIOME_ATAC_GLI3_B4
characteristics.characteristic: Submitter Id
characteristics.text: 36
characteristics.unit: year
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: 409B2
characteristics.characteristic: cell_line
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Cerebral organoids were generated using a whole brain organoid differentiation protocol (Lancaster et al. 2013 https://doi.org/10.1038/nature12517). To generate Cas9 mediated GLI3 KO cell lines, two days prior to lipofection, iPSC media was supplemented with 2 µg/ml doxycycline (Clontech, 631311) to induce Cas9n expression. Two guides were designed using the Broad Institute’s CRISPR design tool (http://crispr.mit.edu/). The following guide pair was selected: ACAGAGGGCTCCGCCACGTGTGG, CCGCGGGACGGTGTTTGCCATGG. The Alt-R CRISPR-Cas9 System (IDT) was used for guide delivery with lipofection according to the manufacturer's protocol. To form the crRNA:tracrRNA complex in a 3 µM final concentration for each guide complex, 1.5 µl of each guide crRNA was combined with 3 µl tracrRNA and 44 µl nuclease-free water. For the reverse transfection, 1.5µl of the crRNA:tracr complex mix and 0.75 µl RNAiMAX (Invitrogen, 13778075) were diluted in 47.75 µl OPTI-MEM (Gibco, 1985-062) for each replicate and incubated for 20 minutes at RT in a well of 96-well plate coated with Matrigel (Corning, 35248). During incubation, ~70% confluent cells were detached with TryplE (Gibco, 12605010), centrifuged and resuspended in 1ml mTeSR with Y-27632 (final concentration 10µM, STEMCELL, 72302). After complex incubation, cells were diluted 30 or 60 times in 100 µl mTeSR with Y-27632 (STEMCELL, 72302) and 2 µg/ml doxycycline (Clontech, 631311) and the cell suspension was added to a well containing the transfection complexes. After 24h media was replaced with mTeSR1 media and cells were allowed to recover for 72h. 70% confluent wells were used for further processing after 72 hours. Cells were passaged as single cells in a Matrigel-coated (Corning, 35248) 6-well plate in mTeSR media with 1:200 P/S (Gibco, 15140122) and Y-27632 (STEMCELL, 72302). Low amounts of cells were plated per well to avoid the fusion of colonies. Media was changed daily and Y-27632 was added for the first 72 hours to prevent apoptosis of the single cells. When colonies were apparent, single colonies were picked by scraping with a 10 microliter pipette tip. 2/3 of the cell suspension was plated in a single well of a Matrigel-coated 96-well plate in mTeSR1 supplemented with 1:200 P/S and Y-27632. The other portion of the cell suspension was pelleted and used for validation of frameshift mutations by sequencing. Validated clones were expanded, cryopreserved and karyotyped. Single cell suspensions were acquired by dissociation of the organoids with a papain-based dissociation (Miltenyi Biotec, 130-092-628). Nuclei were isolated according to the protocol provided by 10x genomics (Demonstrated protocol CG000365, Rev B) with a lysis time of 3 min. Combined scRNA-seq and scATAC-seq libraries were generated with the Chromium Single Cell Multiome ATAC + Gene Expression kit according to the manufacturer's protocol. Combined scRNA-seq and scATAC-seq libraries were generated with the Chromium Single Cell Multiome ATAC + Gene Expression kit according to the manufacturer's protocol.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: CRISPR Cas9 mediated GLI3 knockout (29 and 41 bp deletion)
characteristics.characteristic: genetic modification
characteristics.text: inducible Cas9 nuclease
characteristics.characteristic: genotype
characteristics.text: cerebral organoid culture
characteristics.characteristic: growth condition
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: dermis
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: female
characteristics.characteristic: sex
characteristics.text: MULTIOME_ATAC_GLI3_B4
characteristics.characteristic: title