|
| Status |
Public on Oct 13, 2018 |
| Title |
Control Dye Swap [DII_BatchvBatchPCE_1] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Pooled batch PCE fed mixed community sampled three days after starvation
|
| Organism |
Dehalococcoides mccartyi 195 |
| Characteristics |
community: Donna II
community composition: Mixed; DMC population with fermenters and methanogens
sampled time: 3 days
|
| 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) and were fed with 1 µL neat PCE, 40 µL of 100 mM 1,2,3,4-TeCB (Acros Organics) in hexadecane (Sigma Aldrich), 40 µL of 99% pure butyrate (Sigma Aldrich), 100 µL of 50 g/L YE, and 100 µL of vitamin solution. Triplicate samples were taken at 2 and 3 days after feeding.
|
| 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 II © 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 |
Pooled batch PCE fed mixed community sampled three days after starvation
|
| Organism |
Dehalococcoides mccartyi 195 |
| Characteristics |
community: Donna II
community composition: Mixed; DMC population with fermenters and methanogens
sampled time: 3 days
|
| 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) and were fed with 1 µL neat PCE, 40 µL of 100 mM 1,2,3,4-TeCB (Acros Organics) in hexadecane (Sigma Aldrich), 40 µL of 99% pure butyrate (Sigma Aldrich), 100 µL of 50 g/L YE, and 100 µL of vitamin solution. Triplicate samples were taken at 2 and 3 days after feeding.
|
| 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 II © 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.
|
| Description |
Technical replicate 1 of 2
|
| Data processing |
Microarray image analysis was conducted using Agilent Feature Extraction 10.5 Image Analysis Software. The Feature Extraction Software was utilized to perform a background correction and 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.
Geometrically averaged normalized log10 ratio (Cy5/Cy3) representing test/reference and an absolute intensity of the Cy5 channel data generated.
|
| |
|
| Submission date |
Sep 29, 2012 |
| 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 |
GPL16121 |
| Series (1) |
| GSE41231 |
Dehalococcoides mccartyi strain 195's genome-wide transcriptomic response to batch feeding of 1,2,3,4-Tetrachlorobenzene |
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