RNA was isolated from endospore containing cells which were separated from non-spore forming cells using buoyant density gradient centrifugation within an isogenic culture.
Growth protocol
The strain (strain iyfp-abrB-icfp-IIA-amyE; Veening et al., 2004) was grown at 37C and continuously shaken at 245 rpm in sporulation medium. Sporulation medium was prepared as described before (Schaeffer et al., 1965) and contained dehydrated nutrient broth (0.8%), NaOH (0.5 mM), MgSO4 (1 mM), KCl (1 g/l), Ca(NO3)2 (1 mM), MnCl2 (0.01 mM) and FeSO4 (0.001 mM). Cells were harvested after 2 h into stationary growth. To separate endospore containing cells from vegetative cells, the sporulating B. subtilis culture was subjected to Ultravist gradient centrifugation (for details see Veening et al, submitted). After centrifugation, a pellet (mainly containing endospore formers) and a broad toplayer (predominantly containing vegetative cells) were clearly visible. The vegetative cells were removed from the top layer and endospore containing cells were extracted from the pellet. Total RNA was extracted from both fractions and used in subsequent DNA-microarray experiments.
Extracted molecule
total RNA
Extraction protocol
See Lulko et al, 2007. (Lulko AT, Buist G, Kok J, Kuipers OP (2007) Transcriptome analysis of temporal regulation of carbon metabolism by CcpA in Bacillus subtilis reveals additional target genes. J Mol Microbiol Biotechnol, 12, 82-95.)
Label
Cy3
Label protocol
See Lulko et al, 2007. (Lulko AT, Buist G, Kok J, Kuipers OP (2007) Transcriptome analysis of temporal regulation of carbon metabolism by CcpA in Bacillus subtilis reveals additional target genes. J Mol Microbiol Biotechnol, 12, 82-95.)
RNA was isolated from vegetative cells which were separated from endospore forming cells using buoyant density gradient centrifugation within an isogenic culture.
Growth protocol
See Channel 1.
Extracted molecule
total RNA
Extraction protocol
See Lulko et al, 2007. (Lulko AT, Buist G, Kok J, Kuipers OP (2007) Transcriptome analysis of temporal regulation of carbon metabolism by CcpA in Bacillus subtilis reveals additional target genes. J Mol Microbiol Biotechnol, 12, 82-95.)
Label
Cy5
Label protocol
See Lulko et al, 2007. (Lulko AT, Buist G, Kok J, Kuipers OP (2007) Transcriptome analysis of temporal regulation of carbon metabolism by CcpA in Bacillus subtilis reveals additional target genes. J Mol Microbiol Biotechnol, 12, 82-95.)
Hybridization protocol
See Lulko et al, 2007. (Lulko AT, Buist G, Kok J, Kuipers OP (2007) Transcriptome analysis of temporal regulation of carbon metabolism by CcpA in Bacillus subtilis reveals additional target genes. J Mol Microbiol Biotechnol, 12, 82-95.)
Scan protocol
See Lulko et al, 2007. (Lulko AT, Buist G, Kok J, Kuipers OP (2007) Transcriptome analysis of temporal regulation of carbon metabolism by CcpA in Bacillus subtilis reveals additional target genes. J Mol Microbiol Biotechnol, 12, 82-95.)
Description
See Lulko et al, 2007. (Lulko AT, Buist G, Kok J, Kuipers OP (2007) Transcriptome analysis of temporal regulation of carbon metabolism by CcpA in Bacillus subtilis reveals additional target genes. J Mol Microbiol Biotechnol, 12, 82-95.)
Data processing
Two biological replicates were performed and the DNA-microarray slides contained two probes of each ORF. Thus, maximally 4 datapoints for each gene were obtained. It should be noted that due to the timely centrifugation treatment, RNA quality isolated from the recovered cells was compromised, indicating that the obtained microarray data will not completely reflect the true mRNA state of both subpopulations. Also, only a relatively small amount of signal (transcripts) could be detected for the endospore containing fraction. Because of this, standard microarray data analyses could not effectively be used on these datasets. Therefore, we undertook the following approach: raw data (as submitted here) was subjected to MicroPrep analyses (van Hijum et al., 2003, MicroPreP: a cDNA microarray data pre-processing framework. Appl Bioinformatics, 2, 241-244.) (without a Lowess normalization) and genes with a coefficient of variance lower than 60% were taken into account for further analyses. These microprep data and data used for subsequent FIVA analyses (Blom et al 2007) are included in the supplemental excel file.
