|
| Status |
Public on Mar 20, 2013 |
| Title |
hfRPESeq072611 |
| Sample type |
SRA |
| |
|
| Source name |
Retinal Pigment Epithelium
|
| Organism |
Homo sapiens |
| Characteristics |
origen of cultured cells: RPE isolated from 16 weeks gestation human fetus
transepithelial electrical resistance: 730 ± 10 ohm*cm^2
peak transepitheilal electrical resistance (ksr only): N/A
|
| Growth protocol |
RPE was differentiated and isolated from human embryonic stem cell lines H1 and H9 (three independent isolates from each strain) and compared to RPE that was isolated from 16-week gestation human fetuses (two fetuses) and expanded in primary culture. The RPE was cultured on Transwell filters and adapted to a serum-free medium (Peng et al., Invest. Ophthalmol. Vis. Sci. 52:1392-1403, 2011). When the transepithelial electrical resistance became stable, total RNA was isolated for making sequencing library.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Libraries were prepared according to Illumina’s mRNA Sequencing Sample Preparation Guide for mRNA-Seq 8 Sample Prep Kit (Cat# RS-100-0801) for illumina Genome Analyzer II and TruSeq RNA Sample Preparation Guide for TruSeq RNA Sample Prep Kit v2 (Cat RS-122-2001) for HiSeq 2000. Briefly, poly-A containing mRNA molecules were purified from 10ug of total RNA using poly-T oligo-attached magnetic beads then fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNaseH. These cDNA fragments then go through an end repair process using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3’ to 5’ exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. These products are then purified and enriched with 15 cycles of PCR to create the final cDNA library. Library fragments of 150 to 350 bp (insert plus adaptor and PCR primer sequences) were isolated from 2% E-Gel EX Gel (Invirotgen #G4020-02). The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
hfRPESeq072611
|
| Data processing |
Basecalls performed using CASAVA version 1.7.0 and 1.8.1
Sequenced reads from the sequencing run were imported into the public Galaxy platform, a free online bioinformatics interface available at http://main.g2.bx.psu.edu. The sequences were aligned against the hg19 reference genome using the Tophat for Illumina (version 1.5.0) with default parameters.
Transcripts were assembled using Cufflinks (version 0.0.5) with quartile normalization and bias correction.
Fold change in transcript expression were analyzed using Cuffdiff (version 0.0.5) with false discovery rate of 0.05, minimum alignment count of 100, quartile normalization, and bias correction.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files for RPKM values of each sample and tab-delimited text files for fold changes between two compared cell types
|
| |
|
| Submission date |
Mar 18, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Mei Zhong |
| E-mail(s) |
mei.zhong@yale.edu
|
| Phone |
203-737-6203
|
| Fax |
203-785-4305
|
| URL |
http://stemcell.yale.edu/index.aspx
|
| Organization name |
Yale University
|
| Department |
Stem Cell Center
|
| Lab |
Genomics Core
|
| Street address |
10 Amistad Street
|
| City |
New Haven |
| ZIP/Postal code |
06510 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE36695 |
Comparison of stem-cell derived retinal pigment epithelia (RPE) with human fetal retina pigment epithelium |
|
| Relations |
| SRA |
SRX251952 |
| BioSample |
SAMN01983922 |
