|
| Status |
Public on Oct 13, 2018 |
| Title |
DII_PSSPCEvBatchPCE_2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
DET containing DII, Continous Fed PCE 7.3 μmol/(L/hr)
|
| Organism |
Dehalococcoides mccartyi |
| Characteristics |
strain: strain 195 in the Donna II Mixed Community
feed type: Continous
electron acceptor: PCE
electron donor: Butyrate+Yeast Extract
electron acceptor respiration rate (µeeq/(l-hr)): 43.3
batch acceptor feed concentration (µeeq/l): N/A
electron donor/electron acceptor ratio on eeq basis: 4.54
length of experiment (hours): 168
|
| 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.
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| |
|
| 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 |
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