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Sample GSM302514 Query DataSets for GSM302514
Status Public on Jul 25, 2008
Title #11_C9
Sample type genomic
 
Channel 1
Source name Semen donor #11
Organism Homo sapiens
Characteristics Single sperm prepared by flow cytometry
Extracted molecule genomic DNA
Extraction protocol Total RNA extracted using Trizol following manufacturer's instructions. The procedures for multiplex amplification and genotype determination are illustrated in Figure 1. First, multiplex PCR (Fig. 1A) was performed in 30 µL of PCR mix containing 1x PCR buffer (50 mM KCl, 100 mM Tris-HCl at pH 8.3, 1.5 mM MgCl2, and 100 µg/mL gelatin), four dNTPs (200 µM each; Invitrogen), primers (20 nM each) for all SNPs in the multiplex group, 6 units of HotStar Taq DNA polymerase (QIAGEN), and 5 ng of DNA or single sperm. The samples were first heated to 94°C for 15 min to activate the Taq DNA polymerase followed by 40 PCR cycles. Each PCR cycle consisted of 40 sec at 94°C for denaturation and 2 min at 55°C followed by 5 min of ramping from 55°C to 70°C for annealing and extension. A final extension step was carried out at 72°C for 3 min at the end of the 40-th cycle. PCR amplifications were performed with thermal cyclers capable of ramping as slow as 0.01°C/sec. ssDNA was generated in both directions by using the same conditions for multiplex PCR except: (1) 1-2 µL of product from the multiplex PCR was used as templates, (2) only one primer for each SNP; and (3) 45 PCR cycles.
Label Cy5-ddCTP
Label protocol Probes were labeled in 25 µL of labeling solution containing Sequenase buffer), 0.5 units/µL Sequenase (Amersham Pharmacia Biosciences), Cy3-ddATP and Cy5-ddGTP (PE Biosystems) for AG probes, and Cy3-ddUTP and Cy5-ddCTP for CT probes (750 nM each). The reaction was incubated at 70°C for 10 min. The slide was washed under conditions as described above.
 
Channel 2
Source name Semen donor #11
Organism Homo sapiens
Characteristics Single sperm prepared by flow cytometry
Extracted molecule genomic DNA
Extraction protocol Total RNA extracted using Trizol following manufacturer's instructions. The procedures for multiplex amplification and genotype determination are illustrated in Figure 1. First, multiplex PCR (Fig. 1A) was performed in 30 µL of PCR mix containing 1x PCR buffer (50 mM KCl, 100 mM Tris-HCl at pH 8.3, 1.5 mM MgCl2, and 100 µg/mL gelatin), four dNTPs (200 µM each; Invitrogen), primers (20 nM each) for all SNPs in the multiplex group, 6 units of HotStar Taq DNA polymerase (QIAGEN), and 5 ng of DNA or single sperm. The samples were first heated to 94°C for 15 min to activate the Taq DNA polymerase followed by 40 PCR cycles. Each PCR cycle consisted of 40 sec at 94°C for denaturation and 2 min at 55°C followed by 5 min of ramping from 55°C to 70°C for annealing and extension. A final extension step was carried out at 72°C for 3 min at the end of the 40-th cycle. PCR amplifications were performed with thermal cyclers capable of ramping as slow as 0.01°C/sec. ssDNA was generated in both directions by using the same conditions for multiplex PCR except: (1) 1-2 µL of product from the multiplex PCR was used as templates, (2) only one primer for each SNP; and (3) 45 PCR cycles.
Label Cy3-ddUTP
Label protocol Probes were labeled in 25 µL of labeling solution containing Sequenase buffer), 0.5 units/µL Sequenase (Amersham Pharmacia Biosciences), Cy3-ddATP and Cy5-ddGTP (PE Biosystems) for AG probes, and Cy3-ddUTP and Cy5-ddCTP for CT probes (750 nM each). The reaction was incubated at 70°C for 10 min. The slide was washed under conditions as described above.
 
 
Hybridization protocol Hybridization was done with 1x hybridization solution (5x Denhart's solution, 0.5% SDS, 5x SSC, 20 µL of ssDNA/1000 microarray spots) in a Hybridization Chamber (Corning) at 56°C for 2.5-4 h. After hybridization, the slide was washed at 56°C with 1x SSC and 0.1% SDS for 10 min, twice with 0.5x SSC for 30 sec, and twice with 0.2x SSC for 30 sec.
Scan protocol Microarrays were scanned with GenePix 4000B (Axon Instruments)
Description #11_C9_R600G500_50313.gpr
Single sperm from donor#11
Data processing The resulting images were analyzed with either the GenePix Pro (Axon Instruments) or ImaGene (BioDiscovery) software.
 
Submission date Jul 01, 2008
Last update date Jul 24, 2008
Contact name Minjie Luo
E-mail(s) luomj@rci.rutgers.edu
Organization name University of Medicine and Dentistry of New Jersey
Department Microbiology & Molecular Genetics
Lab Honghua Li's Lab
Street address 675 Hoes Lane
City Piscataway
State/province NJ
ZIP/Postal code 08854
Country USA
 
Platform ID GPL7012
Series (1)
GSE11957 Genetic Structure of Duplicated Sequences Revealed by Genotyping Single Sperm

Data table header descriptions
ID_REF
F635 Mean
F532 Mean
F635 norm
F532 norm
F635-B
F532-B
VALUE log ratio representing F635/F532
Call genotype call based on relative F635/F532 signal strength
Dup_call

Data table
ID_REF F635 Mean F532 Mean F635 norm F532 norm F635-B F532-B VALUE Call Dup_call
01_01_01 64928 654 55082 770 50971 1 10.8390191660743 C C
01_01_02 64086 678 54367 799 50257 1 10.8249059288211 C C
01_01_03 64382 735 54619 866 50508 1 10.8298900864769 C C
01_01_04 65095 870 55223 1025 51113 1 10.8417948314525 C C
01_01_05 3260 8769 2765 10336 1 7927 -8.97814659940482 T T
01_01_06 3054 7913 2590 9327 1 6918 -8.84201470360124 T T
01_01_07 3215 8291 2727 9772 1 7364 -8.90442429476029 T T
01_01_08 3662 10055 3106 11852 1 9443 -9.15311291588034 T T
01_01_09 52703 36209 44711 42681 40600 40272 0.00809794442197085 C/T C/T
01_01_10 53414 36323 45314 42815 41203 40407 0.0195142123182353 C/T C/T
01_01_11 52471 37542 44514 44252 40403 41843 -0.0350354194939485 C/T C/T
01_01_12 52217 38643 44298 45550 40187 43141 -0.0709269374570008 C/T C/T
01_01_13 1082 55930 917 65927 1 63518 -11.0590907539077 T T
01_01_14 1069 54070 906 63734 1 61326 -11.0239641260315 T T
01_01_15 1141 53706 967 63305 1 60897 -11.0169431324724 T T
01_01_16 1167 53572 990 63147 1 60739 -11.0143460167272 T T
01_02_01 61182 10944 51904 12900 47793 10491 1.51630342664979 C+/T C+/T
01_02_02 61942 11350 52549 13378 48438 10970 1.48509914421702 C+/T C+/T
01_02_03 62377 11513 52918 13570 48807 11162 1.47532637012176 C+/T C+/T
01_02_04 62270 12320 52827 14522 48716 12113 1.39168303691407 C/T C+/T

Total number of rows: 320

Table truncated, full table size 18 Kbytes.




Supplementary file Size Download File type/resource
GSM302514.gpr.gz 30.4 Kb (ftp)(http) GPR
Processed data included within Sample table

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