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Sample GSM2060787 Query DataSets for GSM2060787
Status Public on Feb 22, 2016
Title CiPEHR_warmed_rep1
Sample type genomic
 
Channel 1
Source name soil microbial metagenome in soil warming group
Organisms Bacteria; Archaea; Eukaryota; Viruses
Characteristics soil origin: Akaska acidic tundra
Treatment protocol snow pile warmed soil
Growth protocol uncultured
Extracted molecule genomic DNA
Extraction protocol Soil DNA was extracted using a PowerMax Soil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol. DNA quality was assessed based on spectrometry absorbance at wavelengths of 230 nm, 260 nm and 280 nm detected by a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies Inc., now NanoDrop Products by Thermo Fisher Scientific). The 260/280 nm absorbance ratios were around 1.8, and 260/230 nm ratios were greater than 1.7. Finally, DNA was quantified with Pico Green using a FLUOstar OPTIMA fluorescence plate reader (BMG LabTech, Jena, Germany) and used for gene array labeling and sequencing library preparation.
Label Cy3
Label protocol For each sample, 1µg DNA was diluted to a volume of 25 μL and was mixed with 20 μL random primers (Invitrogen by Life Technologies, Grand Island, NY, USA), denatured at 99.9°C for 5 min and immediately chilled on ice. Then, 2.5 μL dNTP (5 mM dAGC-TP, 2.5 mM dTTP), 1 μL fluorescent dye Cy-3 dUTP (GE Healthcare UK Limited, Buckinghamshire, UK) and 80 U of the large Klenow fragment (Imer Inc., CA, USA) were added to the denatured DNA. The 50 μL mixture was incubated at 37°C for 6 hours, followed by heating at 95°C for 3 min. Labeled DNA was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Cy-3 dye concentration was measured on a NanoDrop ND-1000 spectrophotometer (NanoDrop Products). The labeled DNA was then dried in a Savant SPD1010 SpeedVac concentrator system (Thermo Fisher Scientific) at 45°C for 45 min.
 
Channel 2
Source name synthesized common oligonucleotide reference standard
Organism synthetic construct
Characteristics sequence: 5′-CCGCACCTCGGACCGCACACAATCGTTTGAGGACGTGTAGCTGTGCTGGC-3′
molecule subtype: single stranded oligonucleotide
Extracted molecule genomic DNA
Extraction protocol Soil DNA was extracted using a PowerMax Soil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol. DNA quality was assessed based on spectrometry absorbance at wavelengths of 230 nm, 260 nm and 280 nm detected by a NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies Inc., now NanoDrop Products by Thermo Fisher Scientific). The 260/280 nm absorbance ratios were around 1.8, and 260/230 nm ratios were greater than 1.7. Finally, DNA was quantified with Pico Green using a FLUOstar OPTIMA fluorescence plate reader (BMG LabTech, Jena, Germany) and used for gene array labeling and sequencing library preparation.
Label Cy5
Label protocol For each sample, 1µg DNA was diluted to a volume of 25 μL and was mixed with 20 μL random primers (Invitrogen by Life Technologies, Grand Island, NY, USA), denatured at 99.9°C for 5 min and immediately chilled on ice. Then, 2.5 μL dNTP (5 mM dAGC-TP, 2.5 mM dTTP), 1 μL fluorescent dye Cy-3 dUTP (GE Healthcare UK Limited, Buckinghamshire, UK) and 80 U of the large Klenow fragment (Imer Inc., CA, USA) were added to the denatured DNA. The 50 μL mixture was incubated at 37°C for 6 hours, followed by heating at 95°C for 3 min. Labeled DNA was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Cy-3 dye concentration was measured on a NanoDrop ND-1000 spectrophotometer (NanoDrop Products). The labeled DNA was then dried in a Savant SPD1010 SpeedVac concentrator system (Thermo Fisher Scientific) at 45°C for 45 min.
 
 
Hybridization protocol The labeled DNA was resuspended in 10 µL hybridization solution containing 2.68 µL sample tracking control (Roche NimbleGen, Inc., Madison, WI, USA), 40% formamide, 25% SSC, 1% SDS, 1.6% Cy3 labeled alignment oligo, 1.6% Cy5-labeled alignment oligo and 2% Cy5-labeled common oligonucleotide reference standard target (NimbleGen). After denaturing at 95°C for 5 min, the mixtures were spun down and kept at 42°C to be deposited onto the glass microarray. GeoChip 4.2 slides were synthesized by NimbleGen in a 12-plex format (i.e., 12 arrays per glass slide). An HX12 mixer (NimbleGen) was affixed onto the array using a Precision Mixer Alignment Tool (NimbleGen). The array was preheated at 42°C in the 12-Bay MAUI Hybridization System (BioMicro Systems, Inc., Salt Lake City, UT, USA) for at least 5 min before the samples were loaded onto the array. Hybridizations were performed on the MAUI Hybridization System at 42°C in the presence of 40% formamide for about 16 hours with mixing. Then the mixers were removed from the slides while soaking in buffer I. The slides were manually washed in buffer I for 120 s, in buffer II for 60 s and in buffer III for 15 s and spun to dry in a NimbleGen slide spinner. All washing buffers were provided by NimbleGen.
Scan protocol The dried microarray was scanned with a laser power of 100% and a photomultiplier (PMT) gain of 100% by a MS 200 Microarray Scanner (NimbleGen) at 532 nm and 635 nm. NimbleScan software version 2.5 (NimbleGen) was used to grid the images. After gridding, every spot (containing one unique probe) on the GeoChip array was fixed into a 7 × 7 pixel square and was adjacent to four blank equal-sized squares (void spaces). The probe signal was then calculated as the average intensity of the center 5 × 5 pixels for each spot, and the background noise signal was the average void intensity for each spot. The signal and background intensity report was generated also in NimbleScan software.
Description AK1H
Data processing Raw data from NimbleScan were submitted to the Microarray Data Manager on our website (http://ieg.ou.edu/microarray/) and analyzed using the data analysis pipeline with the following major steps: (i) Raw signal intensities (Cy3 channel, barcode_position_532.exp.ftr file) on each array were multiplied by a normalization weight I, which is the ratio of the maximum average universal standard intensity (Cy5 channel, barcode_position_635.ctr.ftr) among all the samples divided by the average universal standard intensity of each array; (ii) The signal intensities on each array were further multiplied by a normalization weight II, which is the ratio of the maximum total raw intensity (Cy3 channel) among all the samples divided by the total raw intensity of each array; (iii) Spots with SNR (signal to noise ratio, noise defined as the averaged pixel intensity of the area between two adjacent spots, barcode_position_532.void.ftr file) ≥2 were considered as positive. Otherwise they were treated as negative spots with 0 value (iv) Spots with signal intensity lower than 1000 were not considered as positive and were removed in subsequent analysis; (v) If a probe appeared in only one-third or fewer of the samples in one treatment group (two out of six samples), it was removed from that group before any further analyses; (vi) Signal intensities plus 1 were transformed to their natural logarithm; and (vii) The mean ratio in each sample was calculated by dividing the transformed signal intensity of each probe by the average transformed signal intensity for all detected probes in each sample. Altogether, a total of 48,188 functional gene probes were detected across all samples in this study. Since data could be very sparse with many 0 values, this could change data structure and affect subsequent data analysis. To reduce sparsity, original signal intensity instead of 0 were used for spots with SNR between 1.5 and 2.0.
 
Submission date Feb 12, 2016
Last update date Feb 23, 2016
Contact name Jizhong Zhou
E-mail(s) jzhou@ou.edu
Phone (405) 325-6094
Organization name University of Oklahoma
Department Department of Microbiology and Plant Biology
Lab Institute for Environmental Genomics
Street address 101 David L Boren Blvd
City Norman
State/province OK
ZIP/Postal code 73019
Country USA
 
Platform ID GPL21447
Series (1)
GSE77866 Functional gene diversity of microbial communities sampled in 2010 in CiPHER site, Aklaska

Data table header descriptions
ID_REF
VALUE Normalized signal intensities

Data table
ID_REF VALUE
67540332 0.954544834
146323153 0.89230479
84501125 0.973123576
296394412 1.149119559
195978904 0.956073599
89902628 0.956363079
154508996 1.130849021
119491468 0.923446599
77361354 1.085088854
56964354 0.925533635
120599747 1.030285228
158312217 1.137768499
315590591 1.143948211
239804324 1.238475501
71402608 1.129926812
21221778 1.198602405
119898765 1.228366777
197737627 1.016202007
118034411 0.947802783
60476006 1.111515275

Total number of rows: 48188

Table truncated, full table size 997 Kbytes.




Supplementary file Size Download File type/resource
GSM2060787_521708_A09_532.exp.ftr.gz 3.0 Mb (ftp)(http) FTR
GSM2060787_521708_A09_532.void.ftr.gz 2.0 Mb (ftp)(http) FTR
GSM2060787_521708_A09_635.ctr.ftr.gz 2.7 Mb (ftp)(http) FTR
Processed data included within Sample table

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