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Sample GSM201048 Query DataSets for GSM201048
Status Public on Jun 14, 2007
Title S. coelicolor YSK3225 24h Replicate 2
Sample type mixed
 
Channel 1
Source name S. coelicolor RNA R5- medium 24h Replicate 2
Organism Streptomyces coelicolor
Characteristics Strain: YSK3225
Sample: 24h Replicate 2
Growth protocol S. coelicolor A3(2) strain YSK3225 was maintained on R5 solid agar (Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK) for high density spores. For liquid culture, spores were pre-germinated in 2xYT medium (Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK) and inoculated into R5- liquid medium (Huang, J., C. J. Lih, K. H. Pan, and S. N. Cohen. 2001. Global analysis of growth phase responsive gene expression and regulation of antibiotic biosynthetic pathways in Streptomyces coelicolor using DNA microarrays. Genes Dev 15:3183-92) at a density of ~10exp7 spores/ml. Baffled 2L flasks containing stainless steel coils with 350 ml working volume were used for liquid cultures. Cultures were incubated at 30°C in an orbital shaker at 300 rpm. For measuring cell growth, samples were collected at regular intervals and mixed with one-eighth volume of 2.5 M HCl and then subjected to sonication to break mycelial pellets (Brana, A. F., S. Wolfe, and A. L. Demain. 1986. Relationship between nitrogen assimilation and cephalosporin synthesis in Streptomyces clavuligerus. Arch Microbiol 146:46-51). Absorbance of the resulting solution at 450 nm was then taken as a measure of growth. Proper dilution was made to ensure an OD450 measurement below 1, which is in the linear range of detection.
Extracted molecule total RNA
Extraction protocol Immediately after culture samples were collected, one-fifth volume of an ice-cold stop solution (5% phenol in ethanol) (Bernstein, J. A., A. B. Khodursky, P. H. Lin, S. Lin-Chao, and S. N. Cohen. 2002. Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays. Proc Natl Acad Sci U S A 99:9697-702) was added to the sample volume to prevent RNA degradation. The sample was quickly centrifuged at 4°C and the supernatant was discarded. The remaining sample was then stored at -80°C. To isolate RNA, the frozen pellets were transferred to a mortar and pestle and ground to fine powder in the presence of liquid nitrogen. RLT solution from RNeasy Mini Kit (Qiagen Inc., Valencia, CA) was added before the ground pellets thawed. The remaining steps for RNA isolation were carried out as per manufacturer's instructions. RNA integrity was determined by gel electrophoresis while its quantity and purity were estimated by UV absorbance at OD260 and OD260/OD280, respectively. Total RNA was reverse transcribed to cDNA by SuperscriptII (Invitrogen, Carlsbad, CA) with random hexamers (IDT, Coralville, IA) incorporating aminoallyl-dUTP (Ambion, Austin, TX).
Label Alexa 647
Label protocol The cDNA was then incubated with Alexa 647 (Invitrogen, Carlsbad, CA) for labeling as per manufacturer's instructions.
 
Channel 2
Source name S. coelicolor genomic DNA
Organism Streptomyces coelicolor
Characteristics Strain: M145
Growth protocol Spores for S. coelicolor M145 were generated on Mannitol-Soy flour or R5 agar Cultures for genomic DNA preparation were performed in YEME medium with 0.5% glycine supplement at 30°C until early stationary phase (refer to T Kieser, MJ Bibb, MJ Buttner, KF Chater, DA Hopwood: Practical Streptomyces Genetics: The John Innes Foundation, Norwich, UK; 2000).
Extracted molecule genomic DNA
Extraction protocol Genomic DNA (gDNA) extraction was carried out using Kirby mix procedure as described elsewhere (T Kieser, MJ Bibb, MJ Buttner, KF Chater, DA Hopwood: Practical Streptomyces Genetics: The John Innes Foundation, Norwich, UK; 2000). About 500 µl of 20 µg/µl gDNA was sonicated briefly for 30-40 sec for shearing them to ~500 bp average size (confirmed by gel electrophoresis).
Label Cy3
Label protocol The DNA was labeled with Label IT® Cy3 Labeling Kit (Mirus Bio Corp., Madison, WI) according to suppliers instructions.
 
 
Hybridization protocol The labeled cDNA and gDNA were then mixed and co-hybridized to microarray slides in the presence of 50% formamide. After 16 hours incubation at 50°C, slides were washed and scanned.
Scan protocol Arrays were scanned using ScanArray5000 (Perkin Elmer, Wellesley, MA). Images were analyzed using GenePix (Axon Instruments, Union City, CA) to obtain raw intensity data for each spot. The median fluorescence intensity from each spot was used for all subsequent analysis.
Description No additional description
Data processing A quantile normalization method was employed to normalize these log2 ratios from each hybridization (S Mehra, W Lian, KP Jayapal, SP Charaniya, DH Sherman, WS Hu: A framework to analyze multiple time series data: a case study with Streptomyces coelicolor. J Ind Microbiol Biotechnol 2006, 33:159-72). The resulting log2 ratios were not only used to compare transcript levels of the same mRNA species in different biological samples, but also used as an estimate of the relative abundance of each mRNA species.
 
Submission date Jun 13, 2007
Last update date Aug 14, 2011
Contact name Karthik P Jayapal
E-mail(s) [email protected]
Organization name University of Minnesota
Department Chemical Engineering and Materials Science
Lab Hu lab
Street address 421 Washington Ave SE
City Minneapolis
State/province MN
ZIP/Postal code 55414
Country USA
 
Platform ID GPL4908
Series (1)
GSE8109 S. coelicolor YSK3225 R5- Medium Time-course Study (Culture #2)

Data table header descriptions
ID_REF Identifier corresponding to the ID column of the reference platform
VALUE Normalized average Log2(Cy5/Cy3 Ratio) i.e. (cDNA/gDNA)
Cy5_Sig_Median(1) CH1 cDNA Foreground Median Intensity (replicate 1)
Cy5_Bkd_Median(1) CH1 cDNA Background Median Intensity (replicate 1)
Cy3_Sig_Median(1) CH2 gDNA Foreground Median Intensity (replicate 1)
Cy3_Bkd_Median(1) CH2 gDNA Background Median Intensity (replicate 1)
Cy5_Sig_Median(2) CH1 cDNA Foreground Median Intensity (replicate 2)
Cy5_Bkd_Median(2) CH1 cDNA Background Median Intensity (replicate 2)
Cy3_Sig_Median(2) CH2 gDNA Foreground Median Intensity (replicate 2)
Cy3_Bkd_Median(2) CH2 gDNA Background Median Intensity (replicate 2)

Data table
ID_REF VALUE Cy5_Sig_Median(1) Cy5_Bkd_Median(1) Cy3_Sig_Median(1) Cy3_Bkd_Median(1) Cy5_Sig_Median(2) Cy5_Bkd_Median(2) Cy3_Sig_Median(2) Cy3_Bkd_Median(2)
SCO0001 -1.111 967 82 3054 412 493 98 2127 429
SCO0003 -1.140 871 101 3034 390 1058 120 4281 447
SCO0005 -1.207 780 128 3069 454 911 144 3669 420
SCO0008 -1.276 622 99 3384 338 513 111 1747 329
SCO0009-a -1.535 185 119 924 416 219 130 1214 422
SCO0009-long -1.960 306 143 3415 357 604 107 3596 397
SCO0012 -1.860 392 90 2482 399 233 72 2156 373
SCO0013 -1.009 734 96 2749 571 1325 100 3944 520
SCO0014 -1.691 322 104 1864 447 306 98 2243 315
SCO0015 -0.932 835 103 2890 399 1133 122 3210 422
SCO0016 -1.982 161 110 1398 398 147 95 1255 331
SCO0017 -2.011 238 88 2139 441 307 71 2675 355
SCO0018 -0.522 1805 165 4036 553 2240 130 4815 586
SCO0019 -1.622 210 118 1522 382 326 125 1668 445
SCO0020 -1.817 552 100 3384 387 426 93 3711 438
SCO0021 -1.553 329 121 1902 503 378 125 2077 464
SCO0022 -1.441 379 119 2266 449 549 158 2335 410
SCO0025 -1.739 226 106 1432 335 206 91 1528 347
SCO0026 -1.163 557 102 2098 537 476 99 1846 469
SCO0027 -1.230 712 101 2120 412 333 121 1933 396

Total number of rows: 7629

Table truncated, full table size 363 Kbytes.




Supplementary file Size Download File type/resource
GSM201048.gpr.gz 1.6 Mb (ftp)(http) GPR
Processed data included within Sample table

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