|
| Status |
Public on Dec 13, 2016 |
| Title |
157EV1S (Npl3 IP 42°C) |
| Sample type |
SRA |
| |
|
| Source name |
HKY157
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: HKY157 Cell lysate
stress: heat stress (30 min, 42°C)
ip: Npl3
|
| Treatment protocol |
RNA Co-IP: cell pellets were washed in 1 ml RIP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0,2 % (v/v) Triton X-100, 2 mM DTT, 10 U RiboLockTM RNase Inhibitor (Thermo Scientific)). One volume of the pellet was mixed with 1.5 volumes of RIP buffer, containing EDTA-free Protease inhibitor cocktail (Roche) and one volume of glass beads. Cells were lysed by vigorous mixing for 30 sec at 4 m/s using the FastPrep®-24 machine (MP Biomedicals).Co-immunoprecipitation experiments were performed at 4 °C for 4h by incubating the lysates with Protein G sepharose beads (Amersham Biosciences) bound to monoclonal c-myc (9E10) antibody (Santa Cruz) and GFP-Trap beads (Chromotek). Afterwards beads were washed five times with RIP buffer. Eluate sample was treated with 20 µg proteinase K at 55 °C for 20 min to remove RNA bound proteins.
|
| Growth protocol |
Yeast cells were grown at 25°C in YPD full medium to log phase. Cells were splitted into two parts and collected by centrifugation. Pellets were resuspended in 50mL YPD medium and incubated either at 25°C or 42°C in a petri dish for 30 min. After subsequent UV cross-linking cells were harvested and further porcessed by RNA Co Immunoprecipitation (IP).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Eluates were purified via trizol-chloroform (Ambion® RNA by Life technologies™) extraction. Removal of contaminating DNA was performed using the TURBO DNA-free kit (Ambion® RNA by Life technologies™).
500 ng of total RNA were used as start material for library preparation. Quality and quantity of RIP RNA was determined by using the Fragment Analyzer (Advanced Analytical). We started directly with the fragmentation of the RIP samples (200 bp) and cDNA synthesis. mRNA enrichment or depletion of rRNA was avoided. The samples were prepared with the "TruSeq RNA Sample Prep Kit v2" protocol from Illumina. Libraries were validated by using the Fragment Analyzer for sizing and the QuantiFlur Kit from Promega for library quantitation.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Basecalling and demultiplexing was performed using Illumina CASAVA 1.8.2
Sequences were aligned to the genome reference sequence of Saccharomyces cerevisiae (assembly R64-1-1, obtained from www.ensembl.org) using the STAR software (Dobin et al., 2012; version 2.4.2) allowing for 2 mismatches.
Filtering of unique hits and counting of reads overlapping with exons was conducted with featureCounts (Liao et al., 2014, subread version 1.5.0-p1, Ensembl annotation version 84).
Data was preprocessed in the R/Bioconductor environment (www.bioconductor.org) using the DESeq2 package (Love et al., 2014, version 1.8.2) to yield normalized read counts across all samples.
Only genes with at least 10 raw read counts in any of the samples were used for the downstream analysis.
Genome_build: R64-1-1
Supplementary_files_format_and_content: tab-delimited read counts per gene
|
| |
|
| Submission date |
May 17, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gabriela Salinas |
| E-mail(s) |
Gabriela.Salinas-Riester@medizin.uni-goettingen.de
|
| Organization name |
Universitaetsmedizin Goettingen
|
| Department |
Department of Pathology
|
| Lab |
NGS Integrative Genomics
|
| Street address |
Kreuzbergring 57
|
| City |
Goettingen |
| State/province |
Lower-Saxony |
| ZIP/Postal code |
37075 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13821 |
| Series (1) |
| GSE81542 |
Nuclear mRNA quality control is bypassed for rapid export of stress responsive transcripts [RNA-Seq] |
|
| Relations |
| BioSample |
SAMN05006652 |
| SRA |
SRX1770552 |
