title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of retinal organoids derived from RPGR patient-, control- and mutation-corrected-iPSC at different developmental stages
design: RNA-seq of retinal organoids derived from RPGR patient-, control- and mutation-corrected-iPSC at different developmental stages
library_construction_protocol: At w0, we collected patient-, control-, patient-corrected-iPSC using TRIzol. And at w7, w13, w18, w22, we collected retinal organoids derived from patient-, control and corrected-iPSC. For all samples after week 0, 5 ipsc-derived retinal organoids were used. The procedure for inducing early stages of retinal organoid differentiation was based on a previously described protocol with some modifications (Kuwahara et al., 2015). Human iPSCs (patient-1, 2, control-1 and 2) were dissociated into single cells by TrypLE Select (Life Technologies) supplemented with 0.05 mg/ml DNase I (Roche) and 20 μM Y-27632 (Selleck). Single cells were reaggregated in low-cell-adhesion 96-well plates with V-bottomed conical wells (SumilonPrimeSurface plate; Sumitomo Bakelite) at a density of 12,000 cells per well in differentiation medium containing 10% KSR, 45% Iscove's modified Dulbecco's medium (IMDM, Gibco), 45% Hams F12 (F12, Gibco), Glutamax (Life Technologies), monothioglycerol (450 μM, Sigma), 100 U ml-1 penicillin and 100 μg ml-1 streptomycin (Gibco), supplemented with 20 μM Y-27632 (Sellock), under 5% CO2 at 37°C. The day on which suspension culture was initiated was defined as day 0. On day 6, recombinant human BMP4 (R&D) was added to the culture to a final concentration of 55 ng/ml, and its concentration was diluted in half every 3rd day via changing half of the medium.  On day 18, the aggregates were transferred into petri dish and cut into 2 to 3 pieces using a V-Lance Knife (Alcon Surgical). They were suspended in neural retina medium [DMEM/F12-Glutamax medium (Life Technologies) supplemented with 1% N-2 supplement (Life Technologies), 0.5 μM retinal acid (Sigma), 0.1 mM taurine (Sigma), 10% FBS (Biological Industries), 100 U ml-1 penicillin and 100 μg ml-1 streptomycin (Life Technologies)] under 5% CO2 conditions for long term culture. The medium was changed every 7th day protecting from light exposure, especially after 120 days. Total RNA was extracted from retinal organoids using the RNeasy Mini Kit (Qiagen) and TRIzol reagent (Life Technologies) according to the manufacturer's protocols. A total of 120 µg of RNA from the patient, control and corrected 3-D retinae from a series differentiation times (W0, 7, 13, 18 and 22) were sent for RNA sequencing. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext®UltraTM RNA Library Prep Kit for Illumina®(NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer（5X）. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase（RNase H-）. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H . Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 200-250 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.