Homo sapiens
Protocols: Single cell suspensions were acquired by dissociation of the organoids with a papain-based dissociation (Miltenyi Biotec, 130-092-628). Cerebral organoids were generated using a whole brain organoid differentiation protocol (Lancaster et al. 2013). The iCRISPR-Cas9 line was cultured on matrigel in mTesr1 supplemented with P/S (1:200) and Cas9 was induced 2 days prior to lentiviral transduction by adding 2µg/ml doxycycline. 24 hours later cells were dissociated into single cells with TrypLE and 300.000 cells of the iCRISPR-Cas9 cells were plated in at least 12 wells of matrigel-coated 6-well plates in mTesr1 supplemented with P/S (1:200), Y-27632 (final concentration 5µM) and 2µg/ml doxycycline. 24 hours later cells were transduced with a low multiplicity of infection (MOI) where less than 30% of the cells were GFP+ to ensure that the majority GFP+ cells only received one lentivirus per cells. The viral particles were added to the culture media (mTesr1 supplemented with P/S , Y-27632 and 2µg/ml doxycycline). 24h later, media was exchanged for mTesr1 supplemented with P/S and 2µg/ml doxycycline until 70% confluency was reached. Cells were then sorted with fluorescence activated cell sorting (FACS) for GFP-positive cells to enrich for CROP-seq-vector-positive cells and plated on matrigel-coated plates in mTesr1 supplemented with 100 μg/ml Primocin (InvivoGen, ant-pm-1) and Y-27632 (final concentration 5µM). Single cell RNAseq libraries were generated with the Chromium Single Cell 3' V3 Library & Gel Bead Kit. Single cell encapsulation and library preparation were performed according to the manufacturer's protocol. Single cell RNAseq libraries were generated with the Chromium Single Cell 3' V3 Library & Gel Bead Kit. Single cell encapsulation and library preparation were performed according to the manufacturer's protocol.
ENA-FIRST-PUBLIC
2022-08-18
ENA-LAST-UPDATE
2022-08-18
External Id
SAMEA110472531
INSDC center alias
ETH Zurich, DBSSE
INSDC center name
ETH Zurich, DBSSE
INSDC first public
2022-08-18T00:13:25Z
INSDC last update
2022-08-18T00:13:25Z
INSDC status
public
Submitter Id
E-MTAB-11999:CROPSEQ_org_1
broker name
ArrayExpress
cell_line
409B2
cell_type
fibroblast
common name
human
developmental stage
adult
disease
normal
genetic modification
mosaic perturbations targeted at 20 transcription factors (cropseq)
genotype
doxycycline-inducible Cas9
individual
mixed pool of organoids 1
organism part
skin
sample name
E-MTAB-11999:CROPSEQ_org_1