|
| Status |
Public on Jan 01, 2010 |
| Title |
GAL:NRD1 vs. wild-type (expression profile) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
GAL:NRD1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: Nrd1 mutant
growth protocol: Cultures with strains containing NRD1 under the control of the GAL1 promoter and either pRS415-Nrd1 or the vector alone were switched to media containing 2% glucose for 6hr after initial growth in SC-LEU + 2% galactose.
|
| Growth protocol |
Cells were grown overnight in the indicated medium containing 10ug/ml doxycycline, diluted to OD600 0.003 in fresh medium and grown to an OD of 0.7.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from yeast cells by vortexing with glass beads in the presence of hot acid phenol and cleaned by phenol:chlorophorm extraction followed by ethanol precipitation. After DNaseI (GE Healthcare) treatment to remove residual genomic DNA, 25 ug of total RNA was incubated with random hexamers at 70C for 10 min, incubated at 15C for 30 min and reverse transcribed to single-stranded cDNA for 2 hours at 42C with Superscript II (Invitrogen) in the presence of 6 μg/ml Actinomycin D to prevent antisense artifacts. RNA was subsequently removed by hydrolysis and cDNA was cleaned up and concentrated on MinElute PCR purification columns (Qiagen).
|
| Label |
biotin
|
| Label protocol |
cDNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and DNase I (GE Healthcare) at 37ｰC until the average fragment size was approximately 70 bp. The fragmentation reaction was stopped by incubating at 95C for 10 min. Fragmented samples were biotinylated with Biotin-N6-ddATP (Enzo Life Sciences) using terminal deoxynucleotidyl transferase at 37C for 2 hours.
|
| |
|
| Channel 2 |
| Source name |
Wild-type
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: WT
growth protocol: Cultures with strains containing NRD1 under the control of the GAL1 promoter and either pRS415-Nrd1 or the vector alone were switched to media containing 2% glucose for 6hr after initial growth in SC-LEU + 2% galactose.
|
| Growth protocol |
Cells were grown overnight in the indicated medium containing 10ug/ml doxycycline, diluted to OD600 0.003 in fresh medium and grown to an OD of 0.7.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from yeast cells by vortexing with glass beads in the presence of hot acid phenol and cleaned by phenol:chlorophorm extraction followed by ethanol precipitation. After DNaseI (GE Healthcare) treatment to remove residual genomic DNA, 25 ug of total RNA was incubated with random hexamers at 70C for 10 min, incubated at 15C for 30 min and reverse transcribed to single-stranded cDNA for 2 hours at 42C with Superscript II (Invitrogen) in the presence of 6 μg/ml Actinomycin D to prevent antisense artifacts. RNA was subsequently removed by hydrolysis and cDNA was cleaned up and concentrated on MinElute PCR purification columns (Qiagen).
|
| Label |
Biotin
|
| Label protocol |
cDNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and DNase I (GE Healthcare) at 37ｰC until the average fragment size was approximately 70 bp. The fragmentation reaction was stopped by incubating at 95C for 10 min. Fragmented samples were biotinylated with Biotin-N6-ddATP (Enzo Life Sciences) using terminal deoxynucleotidyl transferase at 37C for 2 hours.
|
| |
|
| |
| Hybridization protocol |
6 μg of biotinylated cDNA was hybridized at 45C for 16 hours, and then washed and stained according to Affymetrix protocol EukGE-WS2v4_450 in an Affymetrix Fluidics Station 450.
|
| Scan protocol |
Arrays were scanned in an Affymetrix 7G scanner.
|
| Description |
GAL:NRD1 vs. wild-type expression profile
|
| Data processing |
Raw data files were normalized with Affymetrix Tiling Analysis Software (TAS) v1.1. Treatment and control samples were quantile normalized in pairs using only perfect match probes with a bandwidth of 20.
The files ending in 'forward.bar' and 'reverse.bar' correspond to normalized signals from the forward and reverse strand, respectively.
|
| |
|
| Submission date |
Aug 07, 2009 |
| Last update date |
Aug 09, 2009 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
[email protected]
|
| Organization name |
Mount Sinai School of Medicine
|
| Department |
Genetics and Genomic Sciences
|
| Lab |
Bakel Lab
|
| Street address |
One Gustave L. Levy Place, Box 1498
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL7070 |
| Series (1) |
| GSE17559 |
The Sen1 Helicase Prevents Genome-Wide Spurious Transcription by Controlling Transcription Termination by RNAPII |
|
