NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM2551779 Query DataSets for GSM2551779
Status Public on Jun 03, 2019
Title ThawGradient-Mi-Core3-55cm
Sample type genomic
 
Channel 1
Source name soil microbial metagenome in Mi site
Organisms Bacteria; Archaea; Eukaryota; Viruses
Characteristics soil origin: Akaska acidic tundra
Treatment protocol untreated natural 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 Mi3D55
Data processing Data normalization was completed on a web-based pipeline (http://ieg.ou.edu/microarray/) as follows. First, the raw probe signals, calculated based on the average pixel intensity of each probe, were adjusted based on control dye intensity and sample set total intensity. Second, signals were filtered by removing the spots with a signal to noise ratio less than 2, and the spots with signal intensity less than 2 times background. Third, probes that generated positive signals in less than 10 % of the samples from a given thaw site (all replicates from within each field site and along the depth profile) were considered low abundance genes and removed from that site for downstream analyses. We identified low abundance genes on a site basis instead of globally to retain functional gene probes unique to one site. Fourth, the probe signals were then normalized to represent relative abundance of genes in each sample. Unless otherwise specified, all gene abundance data reported in this manuscript represent relative abundance estimations based on the normalized signal intensities of probes targeting said gene.
 
Submission date Mar 27, 2017
Last update date Jun 03, 2019
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 GPL23230
Series (1)
GSE97107 Functional gene abundance of tundra soil microbial communities sampled at the EML Thaw Gradient site, AK in May 2004

Data table header descriptions
ID_REF
VALUE Normalized fluorescent signal intensity

Data table
ID_REF VALUE
260614748 12.13913842
193225032 14.10262345
88062454 10.54265942
149927332 10.08212059
302918152 11.74427783
229861703 12.29207661
116748148 12.00894278
227496573 10.7299271
269211903 9.828233122
84043129 11.24477288
116256194 12.23943502
33867146 13.40879019
189218408 10.45407572
115259785 10.51358264
121707184 11.64447908
15964325 10.93143747
15840078 10.94776509
183983051 13.75039763
154275098 10.35250823
149912898 11.71081356

Total number of rows: 46455

Table truncated, full table size 905 Kbytes.




Supplementary file Size Download File type/resource
GSM2551779_507665_A04_532.exp.ftr.gz 3.0 Mb (ftp)(http) FTR
GSM2551779_507665_A04_532.void.ftr.gz 2.0 Mb (ftp)(http) FTR
GSM2551779_507665_A04_635.ctr.ftr.gz 2.7 Mb (ftp)(http) FTR
Processed data included within Sample table

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap