A white single colony of S. pombe cells on a YE plate was inoculated into YES liquid medium to grow to 5x(the 6th power of 10)cell/ml at 30°C.
Extracted molecule
genomic DNA
Extraction protocol
The DNA, isolated from immunoprecipitates, was amplified according to previously described methods (Katou et al., 2003. Nature v424, pp 1078-1083). In the first round of amplification, primer A (GGAATTCCAGCTGACCACCNNNNNNNNN) and the template DNA were incubated in reaction mixture containing Sequenase Ver2.0 T7 DNA polymerase (USB). In the second round of amplification, primer B (GGAATTCCAGCTGACCACC) and the product of the first round were incubated in reaction mixture with Ex Taq polymerase (TAKARA).
Label
Cy5
Label protocol
The amplified DNA, from the immunoprecipitates, was labeled with Cy5-dUTP, using an Oligotailing kit (Roche Diagnostics).
A white single colony of S. pombe cells on a YE plate was inoculated into YES liquid medium to grow to 5x(the 6th power of 10)cell/ml at 30°C.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was isolated from S. pombe using the methods described in Matsumoto et al., 1987, Mol. Cell. Biol. vol.7 pp 4424-4430. Genomic DNA, isolated from nuclei was amplified according to previously described methods (Katou et al., 2003 Nature v424, pp 1078-1083). In the first round of amplification, primer A (GGAATTCCAGCTGACCACCNNNNNNNNN) and the template DNA were incubated in reaction mixture containing Sequenase Ver2.0 T7 DNA polymerase (USB). In the second round of amplification, primer B (GGAATTCCAGCTGACCACC) and the product of the first round were incubated in reaction mixture with Ex Taq polymerase (TAKARA).
Label
Cy3
Label protocol
The amplified DNA, from the whole nuclei was labeled with Cy3-dUTP, using an Oligotailing kit (Roche Diagnostics).
Hybridization protocol
The labeled targets (~100ng) were added to the hybridization solution to hybridize to the DNA probes on the microarray, using Genomic solutions GeneTac Hybridization Station, for four hours at 40°C in Genomic solutions GeneTac Hyb buffer (120µl; including 42% formamide). After hybridization, the slides 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°C for 1min.
Scan protocol
Microarrays were scanned using an ArrayWoRx array scanner, and the acquired data was analyzed by ArrayWoRx software (Applied Precision, Inc.). The fluorescence intensity of each spot circle (150µm diameter) was measured using SoftWorx tracker.
Description
Comparing between genomic DNA (Cy3) and DNA from immunoprecipitates with anti Swi6(Cy5) of wild type haploid.
Data processing
The measured fluorescent intensity, designated by I, was corrected as follows to give a corrected intensity designated by C:C=I-M in case of I>M+2s, C=I*2s/(M+2s) in case of I<M+2s. M and s are the average and standard deviation of I of negative control spots for each wave length, respectively. When I = M+2s, that is C=2s, it was set as the detection limit. When C for either Cy3 or Cy5, or both, was greater than 2s, the values were considered to be effective data. The value r' of the each effective detection spot obtained was calculated as follows:
r'=r-m, r=logR(the base of 2), R=(Ccy5/Ccy3), m is the average of r for the unique region , which is on the outside of cen1.
Submission date
Aug 15, 2007
Contact name
Yuji Chikashige
Organization name
National Institute of Information and Communications Technology
