|
| Status |
Public on Jul 08, 2020 |
| Title |
24H10Z_3 |
| Sample type |
SRA |
| |
|
| Source name |
Fetal RPE- Donor "E"
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: retinal pigmented epithelial (RPE) cells
donor id: hfRPE-110912
plating density: 100,000 cells/cm2
wounding condition: Chronic
time point: 24h
substrate: ECIS 96W1E+
|
| Treatment protocol |
Acute samples were wounded one time (1Z). Chronic samples were wounded once every 24-hours for 10 consecutive days (10Z). Wounds were applied using ECIS Zθ with a wound current of 3000 µA, frequency of 60000Hz, and a wound time of 15 sec. Samples were let to recover for 5-hours, 24-hours, or 8-days after final wound treatment before harvesting with a 1.5mm biopsy punch. Unwounded samples were used as controls (0Z)
|
| 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. ECIS 96W1E+ plates were coated with 10mM cysteine per manufacturer recomendations before coating with 20 ug/m laminin. Fetal cells were plated at 100,000/cm2 and allowed to differentiate for 32-40 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested using NucleoSpin RNA XS kit (Clontech Laboratories)
RNA was converted into cDNA using SMART-Seq v4 Ultra Low Input RNA Kit (Clontech Laboratories). DNA libraries were prepared with Ion Xpress™ Plus gDNA Fragment Library Preparation kit and sequenced by an Ion Proton next-generation sequencer (Thermo Fisher Scientific Inc.).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Description |
Chronic Wound 24H time point
|
| Data processing |
Base-calling was carried out using Torrent Suite and default parameters.
Prior to alighments the shared 10bp 5' bases were trimmed and reads less than 40 bp were discarded. Reads are first mapped using STAR to the human genome hg38. Unmapped reads were then alligned using TMAP (map1,2,&3). The results are then combined into one file using samtools and picard-tools.
Gene-level RNA quantification was determined using Partek Genomics Suite 6.6 and the hg38 RefSeq Transcript annotation. Only reads aligned to exons were considered in the determination of the number of reads per gene.
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) as input, 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 |
Mar 12, 2020 |
| Last update date |
Jul 08, 2020 |
| Contact name |
Lindsay J Bailey-Steinitz |
| E-mail(s) |
ljbailey@ucsb.edu
|
| Organization name |
University of California, Santa Barbara
|
| Department |
MCDB, NRI
|
| Lab |
Coffey
|
| Street address |
6131 Biological Sci Bldg II
|
| City |
Santa Barbara |
| State/province |
CA |
| ZIP/Postal code |
93106 |
| Country |
USA |
| |
|
| Platform ID |
GPL17303 |
| Series (1) |
| GSE146884 |
An in vitro model of chronic wounding and its implication for age-related macular degeneration |
|
| Relations |
| BioSample |
SAMN14366121 |
| SRA |
SRX7901495 |
