PRPF8 CLIP seq for iPSC, kidney organoid, retinal organoid, and retinal epithelium derived from c.6926A>C RPE with Retinitis Pigmentosa patients and CRISPR/Cas9 corrected
PRPF8 CLIP seq for iPSC, kidney organoid, retinal organoid, and retinal epithelium derived from c.6926A>C RPE with Retinitis Pigmentosa patients and CRISPR/Cas9 corrected
Human Subjects Informed consent for research studies, according to the protocols approved by Yorkshire and the Humber Research Ethics Committee (REC ref: 15/YH/0365), was obtained for all study participants. Information on the patient and control participants is provided in Table S1.  Derivation and characterisation of RP13-iPSCs We ascertained three related RP type 13 patients with a PRPF8 c.6926A>C (p.H2309P) heterozygous missense mutation characterised by night blindness in the first decade of life, and one patient with a de novo PRPF8 c.6926A>C heterozygous mutation displaying night blindness at the age of 8 years (Table S1) (Towns et al., 2010). Dermal skin fibroblasts were reprogrammed to iPSCs using a non-integrative RNA-based Sendai virus system (Figure S1A), as described in Buskin et al. 2018. Several clones from each RP13 participant were tested for expression of pluripotent markers (Figure S1B), clearance of transgenes (Figure S1C) and genome stability (Figure S1D). Apart from clone RP13-1A1, which displayed a decreased copy number in the Chr 12p region, all the other clones displayed the expected copy number in the loci that are most susceptible to changes in pluripotent stem cells. Based on these data, one iPSC clone from each participant was selected for further studies and referred to as RP13-1A, 1B, 1C, and 2 as shown in Table S1. All iPSCs were maintained in Matrigel-coated plates with mTeSR™ Plus media and passaged using Versene (EDTA 0.02%) in 1:3 -1: 6 ratio. All cultures were maintained at 37°C, in a humidified environment, with 5% CO2. Cells were cryopreserved with freezing media containing 90% foetal bovine serum (FBS), 10% dimethyl sulfoxide and 10 µM Y26732.  Sendai Virus Clearance assay Total RNA was extracted from 5 × 106 iPSCs using TRIzol® reagent following the manufacturer's instructions. Reverse transcription was carried out using 1 µg of total RNA and the SuperScript cDNA Synthesis Kit following the manufacturer's instructions. Polymerase chain reaction (PCR) was carried out using the cDNA from the reverse transcription reaction with GoTaq DNA polymerase. The PCR products were analysed using 2% agarose gel electrophoresis. All primer details are shown in Table S13.   Detection of pluripotency markers by flow cytometry  iPSCs were dissociated by incubation with Accutase for 5 minutes at 37℃