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Sample GSM2075285 Query DataSets for GSM2075285
Status Public on Dec 01, 2016
Title MOA-RPEB_D32
Sample type SRA
Source name Reprogrammed Fetal RPE-Donor "B"
Organism Homo sapiens
Characteristics donor id: hfRPE-071709
plating density: 80,000 cells/cm2
passage number: 8
culture time: 32 Days
treatment: OTX2-MYCN Transduced Passage 7 RPE, 250 nM A-83-01 (Days 1-7)
substrate: TC Plastic
Growth protocol RPE cells were isolated from fetal donor eyes (Advanced Biosciences Resources, Alameda, CA USA) according to the methods of Hu and Bok [Mol. Vis. 2001, 7:14-19; Methods Mol. Biol. 2010, 652:55-73]. Beyond the initial isolation, all cell culture was carried out using a base medium described by Maminiskis [Invest. Ophthalmol. Vis. Sci.2006, 47:3612-3624]. For 2-3 days post-plating the medium included 15% heat inactivated fetal calf serum. Thereafter, it was reduced to 5%. To generate a working cell bank, primary fetal RPE stocks were expanded approximately 10-fold and these working stocks were designated Passage 0. For routine serial passage, cells were harvested using trypsin digestion and plated at 4,000 cells/cm2. At approximately 80% confluence (every 3-5 days depending on passage number) the cells were enzymatically harvested and re-seeded at 4,000 cells/cm2. For the purpose of transcriptome analysis cells were plated at 80,000 cells/cm2 and harvested after 32 days. Cultures were carried out on laminin-coated porous supports (mouse laminin, Life Technologies, Grand Island, NY USA; Millicell-HA Culture Inserts, EMD Millipore Inc., Billerica, MA USA) or laminin-coated tissue culture plastic as indicated. All cultures were fed every 2-4 days by complete exchange of the medium.
Extracted molecule polyA RNA
Extraction protocol Total RNA was purified using miRNeasy mini-preps (Qiagen Inc., Valencia, CA USA)
PolyA RNA was purified from 1 microgram of total RNA using the Magnetic mRNA Isolation Kit (New England Biolabs, Inc ., Ipswitch MA USA) and mRNA-Seq libraries were constructed using the Ion Total RNA-Seq Kit v2 (Life Technologies, Inc., Grand Island NY USA).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Ion Torrent Proton
Description Passage 7 RPE cells were seeded at 1 X 104 cells/cm2. On day 2, cells were treated with 250 nM A-83-01 and transduced with lentiviral vectors that co-expresses MYCN with Venus, a yellow fluorescence protein, (MOI = 1) and OTX2 with Cerulean, a blue fluorescence protein, (MOI = 0.5). After 7-14 days, Venus and Cerulean double-positive cells were enriched by fluorescence activated cell sorting. The sorted cells were then plated in basal medium supplemented with 250 nM A-83-01. After 32-40 days the cells were harvested by trypsin digestion and pigmented cells were isolated by cell sorting based on an increase in the side scatter to forward scatter ratio. The resulting pigmented MYCN/OTX2/A-83-01 reprogrammed cells (MOA-RPE) were designated as being passage 8.
Data processing Base-calling was carried out using Torrent Suite and default parameters
Read alignment was accomplished using a 2-stage pipeline. The ten 5-prime bases from each sequence were trimmed using FASTX Trimmer 0.0.14. Sequences with a remaining length of 50 bases or greater were then aligned to the human genome (hg38 + 45S-RNA and 5S-RNA bait sequences) using TopHat 2.0.12 (Bowtie2 2.2.3) and settings optimized for variable read-length Ion Torrent sequences: read-mismatches = 4; read-gap-length = 4; read-edit-dist = 8; max-multihits =1; b2-i = S,1,1.15; b2-mp=6,2; b2-rdg = 5,2; and b2-rfg=5,2. Any unmapped sequences were passed to TMAP 3.4.1 and aligned using 5-prime and 3-prime soft-clipping (g = 0). The resulting TopHat and TMAP alignments were merged into a single alignment file.
Gene-level RNA quantification was determined using Partek Genomics Suite 6.6 and the hg38 RefSeq Transcript annotation (10/17/14). Only reads aligned to exons were considered in the determination of the number of reads per gene. In the case where there were two sequencing runs for the same condition (RPEC_P0_D32 and ARPE19-2) the alignments results were combined prior to quantification. Any fractional read count values were rounded to the nearest integer.
Protein coding RNA gene level results were normalized using the trimmed mean of the M-values (TMM) method of Robinson and Oshlack [Genome Biology 2010, 11:R25] as implemented by “edgeR”. Using the raw integer read counts for protein coding genes (HUGO Gene Nomenclature Database, 4/13/2015) as input and selection of genes with at least one count per million reads in 3 or more samples, the normalization factor (norm.factor) and total number of mRNAs reads for each sample (lib.size) were determined. The final processed normalized data for each gene is expressed as reads per million aligned reads (RPM) where RPM = (raw read count * 1 x 106) /(norm.factor * lib.size) .
Genome_build: hg38
Supplementary_files_format_and_content: Tab-delimited text; Annotations, mRNA gene-level raw read counts (Reads) and TMM-normalized read counts per million mRNA alignments (RPM) for all protein coding genes
Submission date Feb 29, 2016
Last update date May 15, 2019
Contact name Monte J. Radeke
Phone 805-893-3695
Organization name University of California, Santa Barbara
Department Neuroscience Research Institute
Lab Center for the Study of Macular Degeneration
Street address Neuroscience Research Institiute
City Santa Barbara
State/province CA
ZIP/Postal code 93106-5060
Country USA
Platform ID GPL17303
Series (1)
GSE78740 Restoration of mesenchymal RPE by transcription factor-mediated reprogramming
BioSample SAMN04521073
SRA SRX1604898

Supplementary file Size Download File type/resource
GSM2075285_MOA-RPEB_D32.txt.gz 493.8 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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