Samples taken after 3 h of fermentation were immediately treated with RNAprotect (Qiagen, Hilden, Germany). Therefore, 10 g of sourdough was mixed with 40 ml of RNAprotect that was 2:1 diluted with 1 x phosphate-buffered saline (Invitrogen, Carslbad, USA). This mixture was kept at room temperature for 5 min, followed by centrifugation (1,000 g for 5 min). The supernatant was centrifuged for a second time (5,000 g for 15 min) and the resulting pellet was resuspended in 200 µl TE buffer (30 mM Tris-HCl, 1 mM EDTA, pH 8.0), containing 5 U µl-1 of mutanolysin (Sigma-Aldrich) and 50 µg µl-1 of lysozyme (Sigma-Aldrich). This mixture was incubated in a shaking water bath at 37°C for 1 h. According to the RNeasy Mini Kit protocol (Qiagen), 700 µl of RLT buffer plus ß-mercaptoethanol (10 µl ml-1) was added to the lysate, the solution was mixed, and transferred to a tube containing approximately 50 mg of acid-washed beads (Sigma-Aldrich). After vortexing for 5 min and microcentrifugation (16,060 g for 10 s), 850 µl of supernatant was added to 590 µl of 80% (vol/vol) ethanol, and this mixture was applied on the RNeasy Mini column (Qiagen). Manufacturer’s standard instructions were followed from that point on. RNA quality was checked by both measuring the 260/230 and 260/280 nm ratios, using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, USA) and capillary electrophoresis using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA).
Label
Cy5
Label protocol
RNA was linearly amplified using Genisphere SensAmp kit (Genisphere, Hatfield, Pennsylvania, USA) using 200 ng of total RNA. The protocol was followed according to manufacturer's instructions. The amplified RNA (aRNA) was purified using the RNeasy Mini Kit (Qiagen), and the purified aRNA was Cy5 labeled by a reverse transcription reaction as earlier described by Puskas LG, Zvara A, Hackler L, Van Hummelen P: RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques 2002, 32(6):1330-1340.
Samples taken after 3 h of fermentation were immediately treated with RNAprotect (Qiagen, Hilden, Germany). Therefore, 10 g of sourdough was mixed with 40 ml of RNAprotect that was 2:1 diluted with 1 x phosphate-buffered saline (Invitrogen, Carslbad, USA). This mixture was kept at room temperature for 5 min, followed by centrifugation (1,000 g for 5 min). The supernatant was centrifuged for a second time (5,000 g for 15 min) and the resulting pellet was resuspended in 200 µl TE buffer (30 mM Tris-HCl, 1 mM EDTA, pH 8.0), containing 5 U µl-1 of mutanolysin (Sigma-Aldrich) and 50 µg µl-1 of lysozyme (Sigma-Aldrich). This mixture was incubated in a shaking water bath at 37°C for 1 h. According to the RNeasy Mini Kit protocol (Qiagen), 700 µl of RLT buffer plus ß-mercaptoethanol (10 µl ml-1) was added to the lysate, the solution was mixed, and transferred to a tube containing approximately 50 mg of acid-washed beads (Sigma-Aldrich). After vortexing for 5 min and microcentrifugation (16,060 g for 10 s), 850 µl of supernatant was added to 590 µl of 80% (vol/vol) ethanol, and this mixture was applied on the RNeasy Mini column (Qiagen). Manufacturer’s standard instructions were followed from that point on. RNA quality was checked by both measuring the 260/230 and 260/280 nm ratios, using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, USA) and capillary electrophoresis using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA).
Label
Cy3
Label protocol
RNA was linearly amplified using Genisphere's SensAmp kit (Genisphere, Hatfield, Pennsylvania, USA) using 200 ng of total RNA. The protocol was followed according to manufacturer's instructions. The amplified RNA (aRNA) was purified using the RNeasy Mini Kit (Qiagen), and the purified aRNA was Cy3 labeled by a reverse transcription reaction as earlier described by Puskas LG, Zvara A, Hackler L, Van Hummelen P: RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques 2002, 32(6):1330-1340.
Hybridization protocol
Hybridization mixtures were prepared using 60 pmol labeled aRNA in 210 µl hybridization buffer (GE Healthcare) containing 50% (vol/vol) formamide (Sigma-Aldrich). The hybridization mixtures were denatured at 96°C for 3 min, put on ice for at least 5 min, hold at 42°C for 5 min, and subsequently microcentrifuged (16,060 g for 5 min). Hybridizations were performed on the automated hybridization station HS 4800 Pro (Tecan, Mannedorf, Switzerland) at 32°C for 16 h. Post-hybridization washing (automated) was performed as follows: 20 s at 32°C, 20 s at 27°C, and 30 s at 23°C in 1 x SSC with 0.2% (wt/vol) SDS; soaking 30 s at 23°C; 30 s at 23°C in 0.1 x SSC with 0.2% (wt/vol) SDS; soaking 30 s at 23°C; 30 s at 23°C in 0.1 x SSC with 0.2% (wt/vol) SDS; soaking 30 s at 23°C; and 30 s in 0.1 x SSC at 23°C. Slides were dried using isopropanol and air.
Scan protocol
Slides were scanned using the Agilent scanner (Agilent) at 10 micron and images were analysed using ArrayVision v7 (GE Healthcare).
Description
RNA sampled during four 10-day spontaneous wheat sourdough fermentations with daily back-slopping carried out in the laboratory.
Data processing
We apply a background correction, i.e. we subtract the local background for the local foreground intensities. We compute the base 2 log-ratio (ratio of Cy5/Cy3) for each probe and average the log-ratios over the 4 replicates on the array.
Analysis of natural wheat and spelt sourdough ecosystem during a 10-day spontaneous laboratory fermentation
Data table header descriptions
ID_REF
VALUE
We apply a background correction, i.e. we subtract the local background for the local foreground intensities. We compute the base 2 log-ratio (ratio of Cy5/Cy3) for each probe and average the log-ratios over the 4 replicates on the array.
PRES_CALL_CY5
the number of times a gene was called present in the Cy5 channel, i.e. the foreground intensity is above the background intensity plus three times the average of the local standard deviation of the foreground and background pixels. Each clone has four repeats, hence this number varies between 0 and 4
PRES_CALL_CY3
the number of times a gene was called present in the Cy3 channel, i.e. the foreground intensity is above the background intensity plus three times the average of the local standard deviation of the foreground and background pixels. Each clone has four repeats, hence this number varies between 0 and 4
