Illumina NovaSeq 6000 paired end sequencing; CROP-seq of 2 month old brain organoids
CROP-seq of 2 month old brain organoids
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.