|
| Status |
Public on Dec 12, 2014 |
| Title |
set1FLAG_1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Schizosaccharomyces pombe cells, set1FLAG (H3K4me-) mutant
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: set1FLAG (H3K4me-) mutant
|
| Growth protocol |
Logarithmically growing cultures were grown 18 hours to mid-exponential phase (A595 ~ 0.5) in standard rich media (YEA) shaking at 250 RPM at 30° C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10 mL cultures were harvested by centrifuging 2000 RPM for 2 minutes at room temperature, decanted, then snap frozen in liquid nitrogen. RNA was extracted using a hot acid phenol method (Lyne et al., 2003), then purified with Qiagen RNEasy spin columns according to the manufacturer's protocol.
|
| Label |
Alexa Fluor 647
|
| Label protocol |
20 ug of total RNA was reverse transcribed using anchored oligo-dT and labeled with Alexa Fluor 647 (mutant) or Alexa Fluor 555 (wildtype) using the Superscript III Indirect cDNA Labeling Kit (Invitrogen) according to the manufacturer's instructions.
|
| |
|
| Channel 2 |
| Source name |
Schizosaccharomyces pombe cells, wildtype
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: wildtype
|
| Growth protocol |
Logarithmically growing cultures were grown 18 hours to mid-exponential phase (A595 ~ 0.5) in standard rich media (YEA) shaking at 250 RPM at 30° C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10 mL cultures were harvested by centrifuging 2000 RPM for 2 minutes at room temperature, decanted, then snap frozen in liquid nitrogen. RNA was extracted using a hot acid phenol method (Lyne et al., 2003), then purified with Qiagen RNEasy spin columns according to the manufacturer's protocol.
|
| Label |
Alexa Fluor 555
|
| Label protocol |
20 ug of total RNA was reverse transcribed using anchored oligo-dT and labeled with Alexa Fluor 647 (mutant) or Alexa Fluor 555 (wildtype) using the Superscript III Indirect cDNA Labeling Kit (Invitrogen) according to the manufacturer's instructions.
|
| |
|
| |
| Hybridization protocol |
150 ng each of labeled mutant and wildtype cDNA were combined with 2X Hi-RPM Hybridization Buffer and 10X GE Blocking Reagent from the Agilent Gene Expression Hybridization Kit. Combined samples were hybridized to Agilent 4x44k array slides in an Agilent SureHyb chamber at 65° C, 20 RPM for 24 hours. Slides were washed once with 6X SSPE. (Invitrogen)/0.005% N-Lauroylsarcosine (Sigma L7414), once with 0.06X SSPE/0.005% N-Lauroylsarcosine, and once with Agilent Stabilization and Drying solution according to Agilent protocols.
|
| Scan protocol |
Slides were scanned using an Agilent G2565CA Scanner.
|
| Description |
Gene expression profiling in set1FLAG (H3K4me-) mutant vs. wildtype cells
|
| Data processing |
Scanned slides were processed using Agilent Feature Extraction Software (v.10.5.1.1), extraction protocol GE2_105_Jul10. Red and green channel spot and background intensities from Feature Extraction files were processed using the R/Bioconductor limma package (v.3.10.2) using the read.maimages function (source=agilent). log2 fold-changes of mutant vs. wildtype were determined with the normalizeWithinArrays function using the loess normalization method.
|
| |
|
| Submission date |
Nov 14, 2014 |
| Last update date |
Dec 12, 2014 |
| Contact name |
Hugh P Cam |
| E-mail(s) |
hugh.cam@bc.edu
|
| Phone |
6175526851
|
| Organization name |
Boston College
|
| Department |
Biology
|
| Street address |
Higgins Hall Room 355, 140 Commonwealth Avenue
|
| City |
Chestnut Hill |
| State/province |
MA |
| ZIP/Postal code |
02467 |
| Country |
USA |
| |
|
| Platform ID |
GPL6503 |
| Series (1) |
| GSE63301 |
Heterochromatin assembly and transcriptome repression by Set1 in coordination with a class II histone deacetylase |
|
