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| Status |
Public on Dec 07, 2017 |
| Title |
D4.2 |
| Sample type |
SRA |
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| Source name |
iPSC-derived Retina organoids_D4
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| Organism |
Mus musculus |
| Characteristics |
cell type source: Nrl-GFP wild-type (WT) induced pluripotent stem cells (iPSC)
age: D4
tissue/cell type: iPSC-derived Retina organoids
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| Treatment protocol |
Nrl-GFP iPSCs were differentiated into retinal organoids using the modified HIPRO protocol (PMID: 27667917). At differentiation day (D) 0, iPSCs were plated in PrimeSurface low adhesion U-shaped 96-well plate (Wako) at a density of 3000 cells per well in 100 ml retinal differentiation medium constituted by GMEM (Life Technologies), 1x NEAA (Sigma), 1x sodium pyruvate (Sigma) and 1.5%(v/v) knockout serum replacement (Life Technologies). 20 ml diluted Matrigel (120 ml >9.5 mg/ml Matrigel diluted in 900 ml retinal differentiation medium) (Corning) was added to each well at D1. At D7, Retinal organoids were transferred a 100 mm Poly(2-hydroxyethyl methacrylate) (Sigma)-coated petri dish with 10 ml DMEM/F12 with GlutaMAX (Life Technologies) supplemented with 1x N2 supplement (Life Technologies) and 1x PS (Life Technologies). From D10 to D26, retinal organoids were maintained in DMEM/F12 with GlutaMAX (Life Technologies), 1x PS (Life Technologies), 1x N2 supplement (Life Technologies), 1 mM taurine (Sigma), 500 nM 9-cis retinal (Sigma) and 100 ng/ml insulin-like growth factor 1 (Life Technologies). From D26 and onward, 1x NEAA acid (Sigma), 1x B27 supplement without Vitamin A (Life Technologies) and 2%(v/v) FBS (Atlanta Biologicals) were added to the culture. Half-media exchanges were performed every two days. 1x 2-ME was freshly added to media. The cultures were incubated in 5% O2 from D0 to D10 and in 20% O2 from D10 onwards.
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| Growth protocol |
The mouse Nrl-GFP wild-type (WT) induced pluripotent stem cells (iPSC) clone was generated, characterized and maintained as previously described (PMID: 27667917). Briefly, the iPSC clone was reprogrammed from fibroblasts of embryonic day 14.5 WT Nrl-GFP mice, by infection of individual Doxycyclin-inducible lentiviral vectors carrying Oct3/4, Sox2, Klf4, and c-Myc genes. Embryonic stem cell-like colonies were picked and evaluated by morphology, proliferation rate and Nanog expression. The selected Nrl-GFP iPSCs were maintained on feeder cells (Millipore) at 37℃, 5% CO2. Maintenance medium was comprised of Knockout DMEM (Life Technologies), 1x MEM non-essential amino acids (NEAA)(Sigma), 1x GlutaMAX (Life Technologies), 1x Penicillin-Streptomycin (PS) (Life Technologies), 2000 U/ml LIF (Millipore), and 15% ES cell-qualified fetal bovine serum (FBS) (Life Technologies). Media were full changed performed every day, with 1x 2-Mercaptoethanol (2-ME) (Life Technologies) freshly added. Cells were passaged using TrypLE Express (Life Technologies) every two days.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For RNA extraction, 1-2 million induced pluripotent stem cells or 36 D4 organoids were directly homogenized in 0.5 ml TriPure isolation reagent (Roche). Later time points, from D7 and onward, neural retina were dissected from intact retinal organoids (30 organoids at D7, 20 at D10, 12 from D12 to D32) using a moria nickel-plated pin holder (Fine Science Tools) with 0.25 mm-diameter tungsten needles (Fine Science Tools), before being lysed in 0.5 ml TriPure isolation reagent. RNA was extracted following manufacturer’s instructions and the quality was assessed using Agilent 4200 TapeStation system (Agilent).
Directional RNA-seq library construction was performed from 100 ng of high quality RNA (RIN > 8.0) using the Illumina Stranded mRNA Library Prep Kit (Illumina). Library construction from three biological replicates for all time points was performed.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Fastq files were generated from reads passing chastity filter and used for further analysis. Illumina adapter, polyA, and polyT sequence trimming was performed with Trimmomatic v0.36.
Transcript level quantitation was performed using Kallisto v0.42.4 employing a merged transcript cDNA and ncRNA FASTA file downloaded from the Ensembl ftp site. Gene level quantification was generated by summarization of the transcript level quantitation using tximport v0.99.9 with the option “countFromAbundance=lengthScaledTPM”. The gene level count values were TMM normalized and converted to count per million (CPM) via edgeR.
Genome_build: GRCm38.p4 using Ensembl v84 annotation
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| Submission date |
Aug 18, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew J Brooks |
| E-mail(s) |
[email protected]
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| Phone |
301-443-4906
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| Organization name |
NIH
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| Department |
NEI
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| Lab |
NNRL
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| Street address |
6 Center Dr Bldg 6, Rm 303
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE102794 |
Developmental Transcriptome Dynamics of Induced Pluripotent Stem Cell Retina Organoid |
| GSE102795 |
Developmental Transcriptome Dynamics of the Murine Retina and iPSC Retina organoid |
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| Relations |
| BioSample |
SAMN07516062 |
| SRA |
SRX3102536 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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