Human_A017-corrected-w13-2
Homo sapiens
Protocols: 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.
ENA first public
2020-02-01
ENA last update
2018-02-14
External Id
SAMEA104590889
INSDC center alias
Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, State Key Laboratory for Visual Science
INSDC center name
Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, State Key Laboratory for Visual Science
INSDC first public
2020-02-01T04:02:45Z
INSDC last update
2018-02-14T13:57:15Z
INSDC status
public
Submitter Id
E-MTAB-6490:Human_A017-corrected-w13-2
age
24
broker name
ArrayExpress
cell type
induced pluripotent stem cell
common name
human
disease
Retinitis pigmentosa
genotype
CRISPR/Cas9 mediated correction of RPGR exon 14 2bp frameshift mutation
growth condition
organoid culture
organism part
urine
sample name
E-MTAB-6490:Human_A017-corrected-w13-2
sex
male
time
13