|
| Status |
Public on Apr 08, 2024 |
| Title |
MLR2 |
| Sample type |
SRA |
| |
|
| Source name |
hiPS derived RPE monolayers
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Stem cell-derived Retinal Pigment Epithelial Cells
strain: hiPSC EP1
tissue: hiPS derived RPE monolayers
cell type: Retinal Pigment Epithelial Cells
treatment: UNTREATED
|
| Treatment protocol |
The small molecule kinase inhibitors BAY651942 and BMS345541 were reconstituted in DMSO (10 mM and 200 µM stocks), loaded into Echo qualified 384-well polypropylene microplates, and dispensed precisely in multiples of 2.5 nL droplets at the desired concentrations in triplicate using an ECHO 550 acoustic liquid handling system.
|
| Growth protocol |
hiPSC and hES lines were maintained on Matrigel basement membrane matrix coated tissue culture plates with mTeSR1 medium in 5% O2 and 10% CO2 conditions and amplified by clonal propagation using the ROCK pathway inhibitor blebbistatin. For hRPE differentiation, stem cells were plated at a higher density (25,000 cells per cm2) and maintained in mTeSR1 to form a monolayer. The culture medium was replaced with a differentiation medium (DM) for 7 weeks. Differentiating cells were enzymatically dissociated to make single-cell suspension using 0.25% (wt/vol) collagenase IV and resuspended in AccuMAX. Cells were re-plated on to Matrigel basement membrane matrix coated plates and maintained in an RPE medium [70% DMEM, 30% Ham’s F12 nutrient mix, 2% B27-serum-free supplement, 1% antibiotic-antimycotic solution (Invitrogen, Waltham, MA, USA)] for ~3 months to form mature RPE monolayers.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
First-strand cDNA synthesis was performed with 200 ng total RNA using anchored oligo-dT and SuperScript III First-Strand Synthesis with SuperMix. Second-strand cDNA synthesis was performed using RNase H, DNA Polymerase I, and Invitrogen Second Strand Buffer. Double-stranded cDNA was purified using DNA Clean & Concentrator-5.
Library preparation was performed using the Nextera XT DNA Library Preparation Kit. Libraries were cleaned using Agencourt AMPure XP beads according to manufacturer’s instructions. Libraries were evaluated by the High Sensitivity DNA Kit on a 2100 Bioanalyzer. They were then multiplexed and sequenced on an Illumina HiSeq with 50 bp paired-end reads. Reads were aligned to NCBI build 37.2 using Tophat (v2.1.0). Cuffquant and Cuffnorm (Cufflinks v2.2.1) were used to quantify expression levels and calculate normalized FPKM values.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Reads were aligned to the human (NCBI build 37.2) reference genomes using TopHat with default setttings. Gene expression levels were quantified using Cuffquant with default settings. Gene expression levels were normalized across all samples using Cuffnorm with default settings
Assembly: NCBI build 37.2
Supplementary files format and content: CSV file containing FPKM for all samples
|
| |
|
| Submission date |
Apr 04, 2023 |
| Last update date |
Apr 08, 2024 |
| Contact name |
Srinivasa R. Sripathi |
| E-mail(s) |
ssripathi@rfsw.org
|
| Phone |
214-363-3911
|
| Organization name |
Retina Foundation of the Southwest
|
| Lab |
Henderson Ocular Stem Cell Laboratory
|
| Street address |
9600 N Central Expressway Suite 200
|
| City |
Dallas |
| State/province |
Texas |
| ZIP/Postal code |
75231 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE228934 |
IKKβ Inhibition Attenuates Epithelial Mesenchymal Transition of Human Stem Cell-Derived Retinal Pigment Epithelium |
|
| Relations |
| BioSample |
SAMN34069094 |
| SRA |
SRX19866451 |
