|
| Status |
Public on Sep 17, 2010 |
| Title |
CG AP1 Replicate2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
200989 IP-enriched
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
treatment: MMS
|
| Treatment protocol |
When OD600 is 0.8, treat with 0.03% MMS for 1hr. Fpr PDRs treat with fluconazole (4ug/mL) for 2.2hrs (Cg) or 3.1hrs (Sc)
|
| Growth protocol |
Grow YAP samples at OD600 of 0.2 in SC media. For PDR, grow until OD600 is 0.2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed for 20 minutes at room temperature with 1% formaldehyde and subsequently inactivated with glycine for 5 minutes and harvested by centrifugation (3000rcf, for 3 minutes). Cells were washed three times with ice-cold TBS and lysed for 2 hours with acid-washed glass beads in a Vibrax-VXR 2000. Cells were subsequently sonicated for 4 cycles of 20 seconds (+100 seconds rest) at power setting 2 on a Misonex Sonicator 3000 and incubated with Dynabeads M-280 conjugated with anti-TAP antibody (Open Biosystems #CAB1001). Reversal of cross-linking was performed overnight at 65◦C. Enrichment was verified by qPCR comparison of promoter-specific gene enrichment (of a known target) vs. a housekeeping control gene (ScACT1 or CgACT1) and compared to a non-antibody enriched control. The enriched DNA was amplified via the GenomePlex Whole Genome Amplification Kit (Sigma-Aldrich #WGA2X)
|
| Label |
Cy5
|
| Label protocol |
BioPrime Array CGH Genomic Labeling System (Invitrogen #18095011)
|
| |
|
| Channel 2 |
| Source name |
200989 IP-unenriched
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
treatment: MMS
|
| Treatment protocol |
When OD600 is 0.8, treat with 0.03% MMS for 1hr. Fpr PDRs treat with fluconazole (4ug/mL) for 2.2hrs (Cg) or 3.1hrs (Sc)
|
| Growth protocol |
Grow YAP samples at OD600 of 0.2 in SC media. For PDR, grow until OD600 is 0.2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed for 20 minutes at room temperature with 1% formaldehyde and subsequently inactivated with glycine for 5 minutes and harvested by centrifugation (3000rcf, for 3 minutes). Cells were washed three times with ice-cold TBS and lysed for 2 hours with acid-washed glass beads in a Vibrax-VXR 2000. Cells were subsequently sonicated for 4 cycles of 20 seconds (+100 seconds rest) at power setting 2 on a Misonex Sonicator 3000 and incubated with Dynabeads M-280 conjugated with anti-TAP antibody (Open Biosystems #CAB1001). Reversal of cross-linking was performed overnight at 65◦C. Enrichment was verified by qPCR comparison of promoter-specific gene enrichment (of a known target) vs. a housekeeping control gene (ScACT1 or CgACT1) and compared to a non-antibody enriched control. The enriched DNA was amplified via the GenomePlex Whole Genome Amplification Kit (Sigma-Aldrich #WGA2X)
|
| Label |
Cy3
|
| Label protocol |
BioPrime Array CGH Genomic Labeling System (Invitrogen #18095011)
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially.
|
| Scan protocol |
Arrays scanned using Genepix 4000A scanner and GenePix 6.0 software
|
| Description |
none
|
| Data processing |
Background subtraction and LOESS
|
| |
|
| Submission date |
Apr 24, 2009 |
| Last update date |
Jun 26, 2012 |
| Contact name |
Dwight Kuo |
| Organization name |
UCSD
|
| Department |
Bioengineering
|
| Lab |
Trey Ideker
|
| Street address |
9500 Gilman Dr.
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL8477 |
| Series (1) |
| GSE15818 |
Coeevolution of a transcriptional network by compensatory trans and cis mutations |
|
