|
| Status |
Public on May 20, 2015 |
| Title |
Red5-Flag-TEV-protein A(FTP) -2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Protein binding RNA of Red5-FTP
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
protein tag: Tagged strain with Flag-TEV-protein A in C-terminal
strain: p1160
fraction: Protein binding RNA
|
| Treatment protocol |
Total RNAs from the whole cell extract (WCE) were extracted using TRI Reagent (Sigma Aldrich) according to the manufacturer’s instructions. Immunoprecipited (IPed) RNA-protein complexes were eluted from anti-Flag beads after tandem affinity purifications , followed by the phenol/chloroform/isoamyl alcohol extraction of RNAs.
|
| Growth protocol |
Flag-TEV-protein A (FTP)-tagged strains were grown in YEA at 30°C to OD 1.8-2.2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The IPed RNAs and WCE RNAs were then treated with Turbo DNase (Life Technologies) to remove potential DNA contamination. Reverse transcription was performed using the SuperScriptTM Indirect cDNA labeling system (Life Technologies) with random hexamers.
|
| Label |
Cy5
|
| Label protocol |
The cDNAs from IPed and WCE sample were labeled with Cy5 and Cy3 (GE Healthcare)
|
| |
|
| Channel 2 |
| Source name |
Total RNA of Red5-FTP
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
protein tag: Tagged strain with Flag-TEV-protein A in C-terminal
strain: p1160
fraction: total RNA
|
| Treatment protocol |
Total RNAs from the whole cell extract (WCE) were extracted using TRI Reagent (Sigma Aldrich) according to the manufacturer’s instructions. Immunoprecipited (IPed) RNA-protein complexes were eluted from anti-Flag beads after tandem affinity purifications , followed by the phenol/chloroform/isoamyl alcohol extraction of RNAs.
|
| Growth protocol |
Flag-TEV-protein A (FTP)-tagged strains were grown in YEA at 30°C to OD 1.8-2.2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The IPed RNAs and WCE RNAs were then treated with Turbo DNase (Life Technologies) to remove potential DNA contamination. Reverse transcription was performed using the SuperScriptTM Indirect cDNA labeling system (Life Technologies) with random hexamers.
|
| Label |
Cy3
|
| Label protocol |
The cDNAs from IPed and WCE sample were labeled with Cy5 and Cy3 (GE Healthcare)
|
| |
|
| |
| Hybridization protocol |
Hybridization, blocking, and washing was performed following the Agilent instructions
|
| Scan protocol |
Scanning of arrays was performed using Agilent DNA Microarray Scanner and Agilent Scan control software (v A.8.4.1.)
|
| Data processing |
Agilent Feature Extraction Software (v 10.7.3.1) Protocol: GE2_107_Sep09 with modifications; Background Substraction Method - Local Background; Detrend on Replicates Only - False; Robust Neg Ctrl Stats? - True; Choose universal error, or the most conservative - Use Universal Error Model; Dye Normalization Probe Selection Method/Variable Rank Tolerance - True; Max Number Ranked Probes - -1; Normalization Correction Method - Lowess Only; Spikein Target Used - False
p-value filtering (p<=0.05) and background filtering (BG=median of negative control probes for green channel and red channel, probes with gProcessedSignal < 2x gBG AND rProcessedSignal < 2x rBG are filtered out) Filtered probes value set to 1
|
| |
|
| Submission date |
Jan 14, 2015 |
| Last update date |
May 20, 2015 |
| Contact name |
Tamas Fischer |
| E-mail(s) |
[email protected]
|
| Organization name |
The Australian National University
|
| Department |
The John Curtin School of Medical Research
|
| Lab |
Fischer group
|
| Street address |
Building 131, Garran Rd
|
| City |
Canberra |
| State/province |
ACT |
| ZIP/Postal code |
2601 |
| Country |
Australia |
| |
|
| Platform ID |
GPL19654 |
| Series (2) |
| GSE64980 |
The fission yeast MTREC complex targets CUTs and unspliced mRNAs to the nuclear exosome [RIP-ChIP] |
| GSE64992 |
The fission yeast MTREC complex targets CUTs and unspliced mRNAs to the nuclear exosome |
|
