|
| Status |
Public on Feb 16, 2018 |
| Title |
No Treatment poly, rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Human skin
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: N-TERT
cell type: keratinocytes
treatment: 0 J/m2 UVB + 8hr incubation
fraction: Polysome
|
| Treatment protocol |
UVB irradiation of N-TERT keratinocytes was carried out using Philips FS20T12 UVB broadband light sources as described previously (Lewis et al., 2010). A IL1700 radiometer and a SED240 UVB detector (International Light, Newburyport, MA) were used to measure UVB intensity prior to each experiment using a distance of 8cm from the light source to the culture dish. Cells were always irradiated in EpiLife media, which eliminates any UVC wavelengths, followed by normal incubation settings (37°C and 5% CO2).
|
| Growth protocol |
N-TERT keratinocytes (Dickson et al., 2000) were cultured in EpiLife media (Invitrogen, Carlsbad, CA) supplemented with human keratinocyte growth supplement (HKGS, Invitrogen) and 1000U Penicillin-Streptomycin (Roche, Indianapolis, IN).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol followed by phenol/chloroform extraction. The concentration and quality of total RNA samples were first assessed using Agilent 2100 Bioanalyzer. A RIN (RNA Integrity Number) of five or higher was required to pass the quality control.
The samples are first spiked-in with ERCC Mix 1 to no treatment and Mix 2 to all treated. Then five hundred nanograms of RNA per sample were used to prepared dual-indexed strand-specific cDNA library using TruSeq Stranded mRNA Library PrepKit (Illumina). The resulting libraries were assessed for its quantity and size distribution using Qubit and Agilent 2100 Bioanalyzer. One and a half pico molar pooled libraries were sequenced with 2x75bp paired-end configuration on NextSeq500 (Illumina) using NextSeq 500/550 High Output Kit. A Phred quality score (Qscore) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
mRNA-Seq
NT3-poly
Processed data file: Percent_shift_calculations.xlsx
|
| Data processing |
FASTQ format converted using FASTQgroomer (Galaxy version 1.0.4).
Reads mapped to genome using Tophat (Galaxy version 2.1.0).
FPKM calculated using Cufflinks (Galaxy version 2.2.1.0).
Transcript assemblies merged using Cuffmerge (Galaxy version 2.2.1.0).
Differential expression for total RNA performed on total samples using Cuffdiff an Cuffmerge assembly (Galaxy version 2.2.1.3).
Percent shifts for polysome fractions calculated by difference in percent total of polysome fractions between each treatment group. i.e. polysome FPKM for NT divided by the sum of the polysome and monosome FPKMs for that sample. Then subtract NT by UVB for each replicate to get a shift.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: *total_cufflinks.xls: Excel files include FPKMs for the 'total' samples.
Supplementary_files_format_and_content: Percent_shift_calculations.xlsx: Excel file includes FPKMs for the fractionated samples
Supplementary_files_format_and_content: Total_RNA_cuffdiff.xlsx: Excel file includes differential expression for total RNA.
|
| |
|
| Submission date |
Jun 06, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Ann Collier |
| E-mail(s) |
[email protected]
|
| Organization name |
Indiana University School of Medicine
|
| Street address |
635 N Barnhill Dr MS4067
|
| City |
Indianapolis |
| State/province |
IN |
| ZIP/Postal code |
46202 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE99745 |
RNA-Seq of polysome profiling fractions and whole cell lysates of UVB-irradiated N-TERT keratinocytes |
|
| Relations |
| BioSample |
SAMN07200194 |
| SRA |
SRX2887999 |
