A single colony of S. pombe cells on a YPD plate was inoculated into EMM2 liquid medium to grow to 2x(the 6th power of 10)cell/ml at 26C. After two washes in EMM2-N (EMM2 depleted of nitrogen sources), cells were transferred to EMM2-N with the concentration of 2x(the 7th power of 10)cell/ml and further incubated at 26C for 48 hr. During this incubation, most cells divided twice, nearly reached to 8x(the 7th power of 10)cell/ml in the first 6 hr and arrested at G0 state. After 48 hr incubation in EMM2-N, 4 volumes of fresh EMM2 liquid media was added to the culture for the nitrogen replenishment. Cells were collected at 1 hr after the replenishment.
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was isolated by acid phenol methods described Sanger S. pombe post-genomics website, and then polyA-RNA was purified by Oligotex-dT30<super>mRNA purification kit (Takara).
Label
Cy5
Label protocol
The polyA-RNA (3-6microg) was labeled by CyScribe Post-Labeling Kit (Amasham Pharmacia) with Cy5 with Oligo dT primer.
Channel 2
Source name
L972, nitrogen-starved for 48 hrs, arrested at G0 state
A single colony of S. pombe cells on a YPD plate was inoculated into EMM2 liquid medium to grow to 2x(the 6th power of 10)cell/ml at 26C. After two washes in EMM2-N (EMM2 depleted of nitrogen sources), cells were transferred to EMM2-N with the concentration of 2x(the 7th power of 10)cell/ml and further incubated at 26C for 48 hr. During this incubation, cells divided twice and reached to 8x(the 7th power of 10)cell/ml in the first 6 hr and arrested at G0 state.
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was isolated by acid phenol methods described Sanger S. pombe post-genomics website, and then polyA-RNA was purified by Oligotex-dT30<super>mRNA purification kit (Takara).
Label
Cy3
Label protocol
The polyA-RNA (3-6microg) was labeled by CyScribe Post-Labeling Kit (Amasham Pharmacia) with Cy3 with Oligo dT primer.
Hybridization protocol
The labeled targets were added to the hybridization solution to contain an equal amount of the Cy3 and Cy5, and then were hybridized onto DNA on the microarray, using Genomic solutions GeneTac Hybridization Station, for four hours at 40°C in Genomic solutions GeneTac Hyb buffer 120microl (including 42% formamide ). The slides after hybridization were washed in the following sequence: (1) 2xSSC 0.1%SDS at 40°C for 5min, (2) 0.2xSSC at 25°C for 1min, and (3) 0.1xSSC at 25°Cfor 1min.
Scan protocol
Microarrays were scanned using an ArraywoRx arrayscanner, and the acquired data was analyzed by a SoftwoRx software (Applied Precision, Inc., Seattle, WA). Fluorescent intensity in each spot circle (150microm diameter) was measured by Softworx tracker.
Description
Comparing the growth-resuming cells at 1 hr after nitrogen replenishment (Cy5) with cells at nitrogen-starved G0 state (Cy3).
Data processing
Measured fluorescent intensity, I, was corrected as follows to give a corrected intensity, C:C = I-M, (for I>M+2s), C = I*2s/(M+2s), (for I<M+2s) where M and s are an average and a standard deviation of I of negative control spots for each wave length respectively. When I = M+2s, that is C=2s, it was set to be a detection limit. When C for either Cy3 or Cy5 or both were greater than 2s, the values were considered to be effective data. VALUE (Expression ratio r') of the each effective detection spot obtained thus was scaled as follows: r'=r-m, r=logR(base is 2), R=(Ccy5/Ccy3), m is an average of r of all effective detection spots.
Submission date
Sep 21, 2005
Last update date
Jan 23, 2023
Contact name
Atsushi Matsuda
Organization name
National Institute of Information and Communications Technology
