|
| Status |
Public on Aug 25, 2015 |
| Title |
CTRL_polyA_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
HeLa cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HeLa Tet-inducible INTS11 shRNA cell clone
treatment: control
|
| Treatment protocol |
Inducible cell clones were treated with doxycycline 1microgr/ml 72h before collection. EGF stimulation was performed for 20 minutes with 100ng/ml of recombinant EGF. RNA were extracted using QIAGEN Rneasy columns with on column DNAse treatment.
|
| Growth protocol |
Wild-type Hela cells were cultivated in DMEM 10% FBS, starvation was performed for 48h in 0.5% FBS before EGF treatment
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
NEBNext Strand specific RNA-Seq Sample Prep Kit (New England Biolabs, inc.)
RNA-Seq: Genomic DNA and ribosomal RNA was removed with Turbo DNA-free kit and RiboMinus Eukaryote Kit (Life Technologies). The polyA and non-polyA fractions were isolated by running RNA samples three times through the Oligo(dT) Dynabeads (Life technologies) to ensure complete separation. The resulting RNA fractions were subjected to strand-specific library preparation using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs
RNA-Seq: Subcellular fractionation was followed as described (Cell. 2012 Jul 20; 150(2): 279–290). The cell lysate was re-suspended in cold lysis buffer with 0.15% NP-40, and the sucrose buffer was used to isolate nuclei. 50% glycerol buffer and nuclei lysis buffer contains 1M Urea and 1% NP-40, were performed to isolate nucleoplasmic fraction and chromatin-bound RNA fraction. Chromatin-bound RNA was isolated with Trizol protocol
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
molecule: polyA RNA
total RNA was ribosome-depleted and enriched for polyA or nonpolyA using Ambion (Life Tech) fractionation kit
|
| Data processing |
Image analysis: Firecrest (Illumina pipeline 1.9, default parameters)
base calling: Bustard (Illumina pipeline 1.9, default parameters)
alignment (ChIPseq): bowtie 2.1.0 (end-to-end , sensitive))
alignment (RNAseq): tophat 2 (default parameters)
tag density files: RSeQC package was used to generate BigWiggle files
Genome_build: hg19
Supplementary_files_format_and_content: strand specific bigWig, compatible with UCSC Genome Browser, were generated using RSeqQC package and the wigtobigWig script provided by UCSC.
|
| |
|
| Submission date |
Apr 29, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Alessandro Gardini |
| E-mail(s) |
[email protected]
|
| Phone |
305-243-7394
|
| Organization name |
University of Miami
|
| Department |
Sylvester Comprehensive Cancer Center
|
| Lab |
Ramin Shiekhattar
|
| Street address |
1501 NW 10 Avenue
|
| City |
Miami |
| State/province |
FL |
| ZIP/Postal code |
33136 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE68401 |
Enhancer activation during EGF response |
|
| Relations |
| BioSample |
SAMN03573691 |
| SRA |
SRX1013971 |
