ERC000011
ENA-CHECKLIST
2022-08-18T00:13:25Z
ENA-FIRST-PUBLIC
2022-08-18T00:13:25Z
ENA-LAST-UPDATE
SAMEA110472533
External Id
ETH Zurich, DBSSE
INSDC center name
2022-08-18T00:13:25Z
INSDC first public
2022-08-18T00:13:25Z
INSDC last update
public
INSDC status
ERS12570553
SRA accession
E-MTAB-11999:CROPSEQ_org_3
Submitter Id
ArrayExpress
broker name
409B2
cell_line
fibroblast
cell_type
human
common name
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.
description
adult
developmental stage
normal
disease
mosaic perturbations targeted at 20 transcription factors (cropseq)
genetic modification
doxycycline-inducible Cas9
genotype
mixed pool of organoids 3
individual
Homo sapiens
organism
Homo sapiens
organism
skin
organism part
Homo sapiens
scientific_name
CROPSEQ_org_3
title