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Sample GSM626966 Query DataSets for GSM626966
Status Public on Oct 13, 2018
Title DII_BatchPCEvBatchPCE_1
Sample type RNA
 
Channel 1
Source name DET containing DII, Batch Fed PCE 110 μmol/L
Organism Dehalococcoides mccartyi
Characteristics strain: strain 195 in the Donna II Mixed Community
feed type: Batch
electron acceptor: PCE
electron donor: Butyrate+Yeast Extract
electron acceptor respiration rate (µeeq/(l-hr)): N/A
batch acceptor feed concentration (µeeq/l): 660
electron donor/electron acceptor ratio on eeq basis: 15.7
length of experiment (hours): 6
Treatment protocol In brief, 160 mL glass serum bottles were filled with 100 mL of 3-day starved mixed community culture sampled from the main reactor (Donna II). Varying combination of Pressure Lok syringe volumes (10 mL, 5 mL, 2.5 mL, 500 μL, and 100 μL; VICI Precision Sampling) and concentrations of electron acceptor and donor in media were loaded on a Cole Palmer 74900 Syringe Pump to deliver an expected continuous rate. Rates of respiration and electron donor feed are given as conditions.
Growth protocol All subcultures studied in this experiment were 100 mL samples from a maintained 6L mixed community culture (Donna II) previously described (Rahm and Richardson 2006; Rahm and Richardson 2008; Rowe et al. 2009) The culture contains a DET population (near 5×108 cells/mL; 60% of total population on per cell basis) mixed with methanogens and organisms that ferment organic substrates.
Extracted molecule total RNA
Extraction protocol 50 mL of liquid culture samples were centrifuged-at 14190×g. The centrifuged sample was split into 8 individual RNA extractions with each sample following the RNeasy© Mini Kit (Qiagen) extraction previously outlined (Rahm and Richardson 2008). The 8 distinct RNA extractions were recombined on the spin filter before the first RW1 buffer wash.
Label Cy3
Label protocol The Superscript I © DNAse RNA cleanup, amino-allyl cDNA formation, cDNA cleanup, and cDNA labeling with Cy3 or Cy5 followed the method outlined (Waller 2010). The quality and quantity of the RNA was determined using the RNA 6000 Nano assay on an Agilent 2100 bioanalyzer (Agilent Technologies). The quantity of resulting cDNA was determined by using the Quant-IT™ OliGreen® ssDNA Assay Kit (Invitrogen). A common control RNA pool sampled from the main Donna II reactor after 3 days of starvation was labeled with Cy3.
 
Channel 2
Source name DET containing DII, Batch Fed PCE 110 μmol/L
Organism Dehalococcoides mccartyi
Characteristics strain: strain 195 in the Donna II Mixed Community
feed type: Batch
electron acceptor: PCE
electron donor: Butyrate+Yeast Extract
electron acceptor respiration rate (µeeq/(l-hr)): N/A
batch acceptor feed concentration (µeeq/l): 660
electron donor/electron acceptor ratio on eeq basis: 15.7
length of experiment (hours): 6
Treatment protocol In brief, 160 mL glass serum bottles were filled with 100 mL of 3-day starved mixed community culture sampled from the main reactor (Donna II). Varying combination of Pressure Lok syringe volumes (10 mL, 5 mL, 2.5 mL, 500 μL, and 100 μL; VICI Precision Sampling) and concentrations of electron acceptor and donor in media were loaded on a Cole Palmer 74900 Syringe Pump to deliver an expected continuous rate. Rates of respiration and electron donor feed are given as conditions.
Growth protocol All subcultures studied in this experiment were 100 mL samples from a maintained 6L mixed community culture (Donna II) previously described (Rahm and Richardson 2006; Rahm and Richardson 2008; Rowe et al. 2009) The culture contains a DET population (near 5×108 cells/mL; 60% of total population on per cell basis) mixed with methanogens and organisms that ferment organic substrates.
Extracted molecule total RNA
Extraction protocol 50 mL of liquid culture samples were centrifuged-at 14190×g. The centrifuged sample was split into 8 individual RNA extractions with each sample following the RNeasy© Mini Kit (Qiagen) extraction previously outlined (Rahm and Richardson 2008). The 8 distinct RNA extractions were recombined on the spin filter before the first RW1 buffer wash.
Label Cy5
Label protocol The Superscript I © DNAse RNA cleanup, amino-allyl cDNA formation, cDNA cleanup, and cDNA labeling with Cy3 or Cy5 followed the method outlined (Waller 2010). The quality and quantity of the RNA was determined using the RNA 6000 Nano assay on an Agilent 2100 bioanalyzer (Agilent Technologies). The quantity of resulting cDNA was determined by using the Quant-IT™ OliGreen® ssDNA Assay Kit (Invitrogen). A common control RNA pool sampled from the main Donna II reactor after 3 days of starvation was labeled with Cy3.
 
 
Hybridization protocol For each experiment, Cy5 labeled cDNA from the mixed community mRNA pool was hybridized against an aliquot of common control of Cy3 labeled cDNA from 3-day starved culture. The hybridization, washing, and scanning of the microarray samples was performed by the Cornell University Microarray Core Facility (http://cores.lifesciences.cornell.edu/brcinfo/) and followed the methods outlined by the manufacturer (Agilent Technologies). The general procedure mixed 25 μl (~400 ng) of the labeled cDNA sample with 25 μl 2x Gene Expression (GEx) Hybridization Buffer HI-RPM (5), hybridized the sample to the microarray slide at 65° C for 17 hours, and washed with GEx Wash Buffer 1 and 2 at room and elevated (37° C) temperatures.
Scan protocol Scanned with an Agilent Technologies Scanner G2505C with a 5 μm resolution.
Data processing Microarray image analysis was conducted using Agilent Feature Extraction 10.5 Image Analysis Software. The Feature Extraction Software was also utilized to perform a within array modified LOESS normalization between the Cy5 and Cy3 signals, to calculate a log ratio between the Cy5 and Cy3 channels, and to calculate a modified Student t-test p-value between the Cy5 and Cy3 signal distributions.
 
Submission date Nov 19, 2010
Last update date Oct 13, 2018
Contact name Cresten Mansfeldt
E-mail(s) cbm59@cornell.edu
Organization name Cornell University
Street address 220 Hollister Hall
City Ithaca
State/province NY
ZIP/Postal code 14850
Country USA
 
Platform ID GPL11218
Series (1)
GSE25499 Dehalococcoides ethenogenes strain 195 in mixed culture continuous (PSS) PCE and TCE fed versus batch PCE fed

Data table header descriptions
ID_REF
VALUE LogRatio (non_geometrically averaged) (Cy5/Cy3)
LogRatioError
PValueLogRatio
gProcessedSignal
rProcessedSignal

Data table
ID_REF VALUE LogRatioError PValueLogRatio gProcessedSignal rProcessedSignal
9722 2.09E-01 6.44E-02 1.18E-03 6.07E+02 9.83E+02
10702 1.99E-01 6.42E-02 1.89E-03 6.34E+02 1.00E+03
11654 1.98E-01 6.41E-02 2.02E-03 7.67E+02 1.21E+03
12847 1.97E-01 6.41E-02 2.14E-03 6.32E+02 9.94E+02
13951 1.82E-01 6.37E-02 4.22E-03 8.18E+02 1.24E+03
1812 1.65E-01 6.32E-02 9.15E-03 4.71E+03 6.88E+03
3063 1.97E-01 6.40E-02 2.11E-03 5.29E+03 8.32E+03
6202 1.94E-01 6.39E-02 2.37E-03 4.66E+03 7.29E+03
6897 1.70E-01 6.34E-02 7.25E-03 4.70E+03 6.95E+03
7822 1.92E-01 6.39E-02 2.69E-03 4.37E+03 6.80E+03
1047 0.00E+00 7.47E-01 1.00E+00 8.50E+00 2.76E+00
1144 0.00E+00 7.54E-01 1.00E+00 9.09E+00 2.87E+00
13597 0.00E+00 7.46E-01 1.00E+00 7.86E+00 2.57E+00
15046 0.00E+00 7.37E-01 1.00E+00 7.41E+00 2.51E+00
15740 0.00E+00 7.48E-01 1.00E+00 8.31E+00 2.69E+00
295 -1.09E+00 3.20E-01 6.86E-04 3.19E+01 2.61E+00
2234 0.00E+00 7.38E-01 1.00E+00 7.51E+00 2.53E+00
2989 0.00E+00 7.36E-01 1.00E+00 6.98E+00 2.37E+00
4420 0.00E+00 7.45E-01 1.00E+00 8.84E+00 2.90E+00
10977 0.00E+00 7.44E-01 1.00E+00 8.02E+00 2.63E+00

Total number of rows: 15744

Table truncated, full table size 780 Kbytes.




Supplementary file Size Download File type/resource
GSM626966.txt.gz 1.4 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data provided as supplementary file

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