|
| Status |
Public on Jan 01, 2010 |
| Title |
SEN1tetO7 nucleosome profile |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Nucleosomal DNA from SEN1tetO7 yeast
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: R1158
genotype/variation: Sen1 mutant
|
| Treatment protocol |
Histone-DNA interactions were cross-linked by the addition of methanol-free formaldehyde (final concentration of 2%) into the culture for 30 minutes. Glycine was then added to a final concentration of 125mM for 5 minutes to neutralize the cross-linking reaction.
|
| Growth protocol |
Cells were grown overnight in YPD containing 10ug/ml doxycycline, diluted to OD600 0.003 in fresh medium and grown to an OD of 0.7.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were pelleted, washed once with PBS and resuspended in zymolyase buffer with freshly added 10 mM β-mercaptoethanol. Zymolyase was added to a final concentration of 0.25 mg/mL and cells were spheroplasted at 30°C while gently rolling for 30 min. After zymolyase treatment, cells were pelleted at 6000rpm for 15 minutes and resuspended in micrococcal nuclease (MNase) buffer with freshly added 1 mM β-mercaptoethanol and 500 μM spermidine. The crosslinked genomic DNA was digested with different amounts of MNase (0, 25, 50, 75, 100, and 150 U) at 37°C for 30 min, after which reactions were stopped by adding 5% SDS, 50 mM EDTA. The digested samples were then treated with 2 mg/mL proteinase K and incubated at 65°C overnight. DNA was isolated by phenol/chloroform extraction and concentrated via ethanol precipitation. Following RNA digestion with RNase A, samples were run on agarose gel and the sample with the highest proportion of mononucleosomal DNA fragments was used for hybridizations.
|
| Label |
biotin
|
| Label protocol |
DNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and a DNase I mix at 37°C until the average fragment size was approximately 50 bp. The fragmentation reaction was stopped by incubating at 95°C for 10 min. Fragmented samples were biotinylated with Biotin-N6-ddATP using terminal deoxynucleotidyl transferase at 37°C for 2 hours.
|
| |
|
| Channel 2 |
| Source name |
DNAse treated total genomic DNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: R1158
|
| Treatment protocol |
Histone-DNA interactions were cross-linked by the addition of methanol-free formaldehyde (final concentration of 2%) into the culture for 30 minutes. Glycine was then added to a final concentration of 125mM for 5 minutes to neutralize the cross-linking reaction.
|
| Growth protocol |
Cells were grown overnight in YPD containing 10ug/ml doxycycline, diluted to OD600 0.003 in fresh medium and grown to an OD of 0.7.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were pelleted, washed once with PBS and resuspended in zymolyase buffer with freshly added 10 mM β-mercaptoethanol. Zymolyase was added to a final concentration of 0.25 mg/mL and cells were spheroplasted at 30°C while gently rolling for 30 min. After zymolyase treatment, cells were pelleted at 6000rpm for 15 minutes and resuspended in micrococcal nuclease (MNase) buffer with freshly added 1 mM β-mercaptoethanol and 500 μM spermidine. The crosslinked genomic DNA was digested with different amounts of MNase (0, 25, 50, 75, 100, and 150 U) at 37°C for 30 min, after which reactions were stopped by adding 5% SDS, 50 mM EDTA. The digested samples were then treated with 2 mg/mL proteinase K and incubated at 65°C overnight. DNA was isolated by phenol/chloroform extraction and concentrated via ethanol precipitation. Following RNA digestion with RNase A, samples were run on agarose gel and the sample with the highest proportion of mononucleosomal DNA fragments was used for hybridizations.
|
| Label |
Biotin
|
| Label protocol |
DNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and a DNase I mix at 37°C until the average fragment size was approximately 50 bp. The fragmentation reaction was stopped by incubating at 95°C for 10 min. Fragmented samples were biotinylated with Biotin-N6-ddATP using terminal deoxynucleotidyl transferase at 37°C for 2 hours.
|
| |
|
| |
| Hybridization protocol |
Biotinylated samples were hybridized at 45°C 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 |
SEN1tetO7 nucleosome profile
|
| Data processing |
Raw data files were normalized with Affymetrix Tiling Analysis Software (TAS) v1.1 and visualized with the Integrated Genome Browser. Treatment and control samples were quantile normalized in pairs using perfect match probes only with a bandwidth of 20.
|
| |
|
| Submission date |
Aug 09, 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 |
|
