RNA-seq of retinal organoids differentiated from hESCs carrying mutations in the RB1 gene

hESC lines carrying deleterious mutations in the RB1 gene in heterozygous and homozygous state were generated by genome-editing based on CRISPR/Cas9. Parental cell line and genome-edited cell lines were differentiated into retinal organoids for 152 days based on the Protocol published by Döpper et al., Current protocols, PMID: 32956559. Briefly, single cells were reaggregated in presence of dual SMAD and WNT-inhibition; retinal tissue became visible from day 12 onward. BMP4-induction and addition of small molecules CHIR99021 and SU5402 directed differentiation towards retina and retinal pigment epithelium. Long-term differentiation was carried out in the presence of 10% FBS, taurine and retinoic acids. Organoids were collected at indicated time points and either embedded for cryosectioning and immunostaining or frozen at -80°C for RNA preparation.