|
| Status |
Public on Jul 13, 2015 |
| Title |
lkh1-wt_vs_lkh1-as_replicate1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
log-phase cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: lkh1-wt
treated with: 10 µM 3-MB-PP1 for 8hrs
growth phase: log-phase cells
|
| Treatment protocol |
For prp2-1, yeast were grown at permissive 25 ºC to mid-log then shifted to 37C for 1 hr. For 3-BrB-PP1 treated kinase strains, prp4-wt and prp4-as strains were grown to mid-log phase, 10 μM of 3-BrB-PP1 was added for 1 hr. dsk1-wt and dsk1-as strains were treated with 10 μM 3-BrB-PP1 for 8 hrs and harvested at mid-log phase.
|
| Growth protocol |
Yeast strains were grown in rich YES5 medium to mid-log phase (OD600 0.4-0.8) at 30 ºC unless otherwise noted.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were collected by centrifugation and frozen at -80 ºC. Total RNA was isolated using hot phenol extraction as outlined previously. cDNA was synthesized using 500 U Superscript III (Invitrogen) for each reaction with 40 μg total RNA and 4 hr incubation at 42 ºC.
|
| Label |
Cy3
|
| Label protocol |
All of the cDNA from control samples was labeled with Cy3 dyes and all of the experimental samples with Cy5 dyes. Once samples were labeled experiments were continued without interruption and samples were exclusively kept in the dark. Appropriate Cy5 experimental and Cy3 control samples were combined.
|
| |
|
| Channel 2 |
| Source name |
log-phase cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: lkh1-as
treated with: 10 µM 3-MB-PP1 for 8hrs
growth phase: log-phase cells
|
| Treatment protocol |
For prp2-1, yeast were grown at permissive 25 ºC to mid-log then shifted to 37C for 1 hr. For 3-BrB-PP1 treated kinase strains, prp4-wt and prp4-as strains were grown to mid-log phase, 10 μM of 3-BrB-PP1 was added for 1 hr. dsk1-wt and dsk1-as strains were treated with 10 μM 3-BrB-PP1 for 8 hrs and harvested at mid-log phase.
|
| Growth protocol |
Yeast strains were grown in rich YES5 medium to mid-log phase (OD600 0.4-0.8) at 30 ºC unless otherwise noted.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were collected by centrifugation and frozen at -80 ºC. Total RNA was isolated using hot phenol extraction as outlined previously. cDNA was synthesized using 500 U Superscript III (Invitrogen) for each reaction with 40 μg total RNA and 4 hr incubation at 42 ºC.
|
| Label |
Cy5
|
| Label protocol |
All of the cDNA from control samples was labeled with Cy3 dyes and all of the experimental samples with Cy5 dyes. Once samples were labeled experiments were continued without interruption and samples were exclusively kept in the dark. Appropriate Cy5 experimental and Cy3 control samples were combined.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized to Agilent custom 8 x 15K microarrays for 16 hrs at 60 ºC in the dark hybridization with rotation. Arrays were opened while submerged in Agilent Wash buffer I (Agilent) and transferred to a rack and washed in more Wash buffer I while stirring for 1 min. Arrays were then transferred to wash buffer II at 37 ºC for 1 min. Next, the arrays were transferred to acetonitrile for 1 min and finally Stabilization & Drying solution (Agilent) for 30 sec. The arrays were then immediately scanned in an ozone free chamber using a GenePix 4000B scanner (Molecular Devices).
|
| Scan protocol |
Scanning was carried out using Genepix software with 5 μM pixel size, 2 lines to average, and a focus position of 15. In order to get a balance between Cy5 and Cy3 fluorescent signals we varied the ratio of the PMT for each wavelength until they were approximately equal, usually between 400-600. Some spots on the array were then flagged due to over saturation or spotty hydration using GenePix pro 7 software and excluded from analysis.
|
| Description |
Biological replicate 1 of 2: lkh1-wt_vs_lkh1-as
|
| Data processing |
Raw data was normalized using loess as implemented in the R package limma.
|
| |
|
| Submission date |
Oct 28, 2014 |
| Last update date |
Jul 13, 2015 |
| Contact name |
Jesse J Lipp |
| E-mail(s) |
[email protected]
|
| Organization name |
UCSF
|
| Street address |
600 16th Street
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL19357 |
| Series (1) |
| GSE62752 |
Schizosaccharomyces pombe splicing microarray |
|
