|
| Status |
Public on Jun 11, 2019 |
| Title |
paf1∆trf4∆_3 Tiling Array |
| Sample type |
RNA |
| |
|
| Source name |
KY2276 S. cerevisiae grown to log phase in YPD
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: MATa leu2[delta]0 ura3[delta]0 trf4[delta]::CLONAT paf1[delta]::KANMX
genotype: paf1[delta]trf4[delta]
|
| Growth protocol |
Cells were grown in YPD supplemented with 400 µM tryptophan to a cell density of 1.8-2.5 x 107 cells/mL and harvested by centrifugation at 3000 rpm for 3 minutes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction pelleted cells were resuspended in 12 mL AE buffer (50 mM NaOAc pH 5.2, 10mM EDTA) and combined with an equal volume of acid phenol SDS to a concentration of 0.8%. After incubation at 65 ˚C for 10 minutes and centrifugation the aqueous phase was re-extracted first with PCI (25 phenol: 24 chloroform: 1 isoamyl-alcohol) and then chloroform. Extracted RNA was precipitated by addition of an equal volume of isopropanol and a tenth volume of 3M NaOAc pH 5.2 and incubation on ice for 10 minutes. RNA was pelleted by centrifugation, washed with 70% EtOH, dried and resuspended in TE buffer (pH 7.0). All RNA samples used in tiling array analysis were checked by agarose gel electrophoresis.
|
| Label |
biotin
|
| Label protocol |
RNA samples (100 µg total) were DNase treated (Fermentas #EN0521, Waltham, Ma) and purified using an RNeasy kit (Qiagen #74104, Hilden Germany). RNA was reverse transcribed into cDNA in the presence of 6 ng/µL actinomycin D to prevent antisense artifacts (*Perocchi/Steinmetz), fragmented, and labeled with a 3’ biotin tag.
|
| |
|
| Hybridization protocol |
not provided
|
| Scan protocol |
Affymetrix 7G scanner
|
| Description |
Trf4Paf1.Plus.Strand.bedgraph
Trf4Paf1.Minus.Strand.bedgraph
trf4paf1.m.window.20.bedgraph
trf4paf1.p.window.20.bedgraph
paf1trf4.vs.trf4.plus.window.20.bedgraph
paf1trf4.vs.trf4.minus.window.20.bedgraph
all_rna_before_limma.csv
paftrfVStrf.all.fix.csv
downregulated_regions_paf1trf4_vs_trf4_window_20_1.5-fold_annotations.bed
upregulated_in_paf1trf4_vs_trf4_window_20_1.5-fold_annotations.bed
total RNA reverse transcribed into cDNA in the presence of Actinomycin D using both oligodT and random heximers and biotinylated
|
| Data processing |
Data was analyzed using R, Python and Shell commands described in detail in the linked manuscript
Bedgraph files for each strand are provided with and without sliding window (20bp) smoothing. Bedgraph files of the difference between the strains compared in this study are also provided with a 20bp sliding window normalization applied. Calculated averages of probe intensities over gene annotations prior to Limma analysis as well as the Limma analysis results are provided. Finally annotations for differentially regulated transcripts are provided in BED6 format.
*Strand.bedgraph = averaged bedgraph (no smoothing)
*20.bedgraph = bedgraph smoothed with a 20 probe (~160bp) sliding window. There are smoothed average genome-wide bedgraphs and smoothed differential results bedgraphs.
all_rna_before_limma.csv = results file transcripts genes before limma analysis
*all.fix.csv = results file for transcripts after limma analysis
*annotations.bed = results file de novo identified differentially expressed transcripts
|
| |
|
| Submission date |
Dec 20, 2018 |
| Last update date |
Jun 11, 2019 |
| Contact name |
Karen M. Arndt |
| Organization name |
University of Pittsburgh
|
| Department |
Biological Sciences
|
| Lab |
A311 Langley Hall
|
| Street address |
4249 Fifth Avenue
|
| City |
Pittsburgh |
| State/province |
Pennsylvania |
| ZIP/Postal code |
15260 |
| Country |
USA |
| |
|
| Platform ID |
GPL19774 |
| Series (1) |
| GSE122704 |
The Paf1 complex broadly impacts the transcriptome of Saccharomyces cerevisiae |
|
