|
| Status |
Public on Jan 08, 2018 |
| Title |
B02-3-RNA-MEF-ICOS-CDS-noDox |
| Sample type |
SRA |
| |
|
| Source name |
Mouse Embryonic Fibroblasts
|
| Organism |
Mus musculus |
| Characteristics |
strain: Rc3h1-2-/-
clip antibody: none
tissue: Mouse Embryonic Fibroblasts
|
| Treatment protocol |
For doxycycline (1µg/mL) treatment cells were treated for 14 hours. For 4' OH-tamoxifen (0.3µM) treatment cells were treated for 5 days. For mepazine (20µM) treatment cells were treated for 3 hours. For ribosome profiling cells were treated with cycloheximide 100µg/ml for 1 minute.
|
| Growth protocol |
MEF cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 10 mM HEPES, pH 7.4 at 37°C in 10% CO2. T cells were cultured in DMEM medium supplemented with 10%FBS, 1x non-essential amino acids, 10 mM HEPES, pH7.4, 50 µM β-mercaptoethanol at 37°C in 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For Ribosome profiling lysates were clarified by centrifugation to remove nuclei and treated with ARTSeq Nuclease for 45min or left untreated. RNA fragments protected by the ribosomes and total RNA from lysates were extracted with acid phenol:chloroform:isoamyl alcohol (125:24:1, pH 4.5). Total RNA from T cells were directly extracted from cells using TRIzol. For PAR-CLIP lysates were clarified by centrifugation and Roquin-RNA-complexes were isolated with the 3F12 antibody. After proteinase K digestion RNA fragments were isloated by phenol-chloroform extraction.
Libraries of ribosome protected fragments were peprared according to the ARTSeq Ribosome Profiling Kit for mammalian cells (Illumina). RNA libraries from MEF cells were prepared according to the Encore Complete RNA-Seq DR Multiplex Systems (NuGEN Technologies). RNA libraries from CD4+ T cells were prepared according to the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech) and Nextera XT DNA Sample Preparation Kit (Illumina). PAR-CLIP libraries were prepared according to the small RNA protocol from Hafner et al., Methods 2012.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
mef_dox_rnaseq_counts.csv
|
| Data processing |
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to the mouse transcriptome based on RefSeq (RNA-seq and Ribo-seq) using segemehl v0.1.7-411 with parameters --accuracy 90. PAR-CLIP reads were analyzed using CLIPZ (http://www.clipz.unibas.ch)
Transcript counts were calculated based on uniquely mapped reads. For Ribo-seq, only the reads mapping to the CDS were considered.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include read counts for each sample (RNA-seq and Ribo-seq), as well as Roquin-bound clusters (PAR-CLIP).
|
| |
|
| Submission date |
Aug 26, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Joao C Guimaraes |
| E-mail(s) |
joaoguima@gmail.com
|
| Organization name |
University of Basel
|
| Department |
Biozentrum
|
| Street address |
Klingelbergstrasse 50 / 70
|
| City |
Basel |
| ZIP/Postal code |
4056 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE86110 |
Roquin suppresses PI3K-mTOR signaling to control T cell differentiation and Treg effector function |
|
| Relations |
| BioSample |
SAMN05711385 |
| SRA |
SRX2056281 |
