|
| Status |
Public on Jul 31, 2010 |
| Title |
Δisw2 rrp6, Replicate 1 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Δisw2 rrp6
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: congenic to S288C background
genotype: Δisw2 rrp6
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by standard hot acid phenol extraction.
|
| Label |
Cy5
|
| Label protocol |
25 μg total RNA and 12 μg random hexamers were placed in 40 μl H2O. The reaction was incubated at 70°C for 5 min, 25°C for 5 min, and 4°C for 5 min. 0.5 μL of 1 mg ml-1 Actinomycin D, 20 μl of 5X SuperScript III Buffer (Invitrogen), 8 μl of 0.1 M DTT, 1 μl of 40 U μl-1 RNase Inhibitors (Roche), 4 μL of dNTP mix (10 mM dATP, 10 mM dGTP, 10 mM dCTP, 8.5 mM dTTP, and 1.5 mM dUTP), 4 μl of 200 U μl-1 SuperScript III (Invitrogen), and 20 μL H2O were added, and the reaction was incubated at 25°C for 10 min, 48°C for 90 min, and 70°C for 10 min. 2 μl of 0.1 mg ml-1 RNaseA and 2 μl of 5,000 U μl-1 RNaseH (NEB) were added and incubated at 37°C for 30 min. The reaction was stopped by phenol:chloroform extraction and unincorporated random hexamers were removed with a gel filtration spin column. cDNA was ethanol precipitated and the pellet was suspended in 80 μl H2O.
|
| |
|
| Channel 2 |
| Source name |
Input Genomic DNA from WT
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: congenic to S288C background
genotype: WT
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Input genomic DNA was isolated from WT cells with Qiagen Genomic DNA columns according to the manufacturer's protocols.
|
| Label |
Cy3
|
| Label protocol |
Purified genomic DNA was fragmented with DNaseI to an average size ~25-75 bp. Fragmented DNA (30 ng) was heated at 70°C for 5 min. Then, 5 μl Buffer #4 (NEB), 5 μl of 2.5 mM CoCl2 (NEB), 5 μl of 1 mM Cy3-dUTP (GE Healthcare), and 5 μl of 20,000 U ml-1 terminal deoxytransferase (NEB) were added to a total volume of 50 μl. The reaction was incubated at 37°C for 3 hours. 50 μl H2O was added and labeled DNA was purified with a gel filtration spin column and ethanol precipitation. The pellet was suspended in a final volume of 50 μl H2O.
|
| |
|
| |
| Hybridization protocol |
According to NimbleGen manufacturer's suggested protocol.
|
| Scan protocol |
According to NimbleGen manufacturer's suggested protocol.
|
| Description |
AY202_isw2rrp6_4340_319894.2
Biological replicate of isw2 rrp6 Rep 3.
Technical replicate of isw2 rrp6 Rep 2.
|
| Data processing |
Biweight mean-adjusted log2 ratios (Cy5/Cy3) were generated using NimbleScan software (NimbleGen) for each hybridization.
|
| |
|
| Submission date |
Jul 23, 2010 |
| Last update date |
Jul 28, 2010 |
| Contact name |
Toshio Tsukiyama |
| Organization name |
Fred Hutchinson Cancer Research Center
|
| Department |
Basic Science
|
| Lab |
Toshio Tsukiyama
|
| Street address |
1100 Fairview Ave N
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL10725 |
| Series (1) |
| GSE23108 |
Determination of ncRNAs repressed by Isw2 |
|
