|
| Status |
Public on Jun 15, 2015 |
| Title |
GFP_RNA-Seq rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Jurkat T cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: T Cells
cell line: Jurkat
transgene: GFP
|
| Treatment protocol |
To induce expression of GFP:SF and Tat:SF for RNA-Seq and ChIP-Seq the stable cell lines were treated with 1 μg/ml doxycycline for 16 hrs.
|
| Growth protocol |
CD4+ Jurkat T cells (clone E6.1) were cultured in RPMI1640 (HyClone) with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen). Jurkat T-Rex cells were maintained in the same conditions but in the presence of 10 mg/ml Blasticidin as indicated by the manufacturer's instructions (Invitrogen). The derived Jurkat T-Rex clones (GFP bearing the Strep/Flag (SF) epitope and Tat bearing the SF epitope) were selected with 300 mg/ml of zeocin for four weeks and later cultured with 10 mg/ml Blasticidin and 100 mg/ml zeocin (Invitrogen). To generate the stable GFP:SF and Tat:SF expressing cell lines, the parental Jurkat T-Rex was electroporated with 2 mg of Ssp1-linearized pcDNA4\TO vector (Invitrogen) bearing either insert using a nucleofector kit V and a Nucleofector II (Lonza).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-seq, total RNA samples were prepared from biological duplicate samples with TRIzol (Invitrogen). rRNA-depleted total RNA was then prepared using the Ribominus isolation kit (Invitrogen) and used for library preparation using the SOLiD Total RNA-Seq kit (Applied Biosystems).
Briefly, 500 ng of rRNA-depleted RNA was fragmented and adapters ligated before cDNA synthesis. The cDNA was then size selected, amplified, purified with AmpureXP beads (Beckton Coulter) and quality checked on a Bioanalyzer (Agilent). Samples were quantified by qPCR using SOLiD adapter primers per the manufacturer's instructions (Applied Biosystems) and the EZ-Bead system was used to amplify the libraries onto the sequencing beads for high-throughput sequencing on a SOLiD 5500xl instrument (Applied Biosystems).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB 5500xl Genetic Analyzer |
| |
|
| Description |
rRNA-minus total RNA
|
| Data processing |
RNA-Seq reads were mapped and aligned to the reference genome hg19 with TopHat v2.0
Differential expression was detected using Cufflinks v1.3.0 with the default significance threshold (p-value < 0.05)
Genome_build: hg19
Supplementary_files_format_and_content: RNA-Seq: csv generated by Cufflinks
|
| |
|
| Submission date |
Feb 05, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ivan D'Orso |
| E-mail(s) |
ivan.dorso@utsouthwestern.edu
|
| Phone |
214-633-1374
|
| Organization name |
UT Southwestern Medical Center
|
| Department |
Microbiology
|
| Lab |
NL3.110A
|
| Street address |
5323 Harry Hines
|
| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-9048 |
| Country |
USA |
| |
|
| Platform ID |
GPL16288 |
| Series (2) |
| GSE65688 |
Tat controls RNA Polymerase II and the epigenetic landscape to precisely rewire cellular transcriptional programs (RNA-Seq) |
| GSE65689 |
Tat controls RNA Polymerase II and the epigenetic landscape to precisely rewire cellular transcriptional programs |
|
| Relations |
| BioSample |
SAMN03331666 |
| SRA |
SRX867204 |
