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Sample GSM432738 Query DataSets for GSM432738
Status Public on Jul 29, 2009
Title orr_weaver_salivary_2 extraction2_array1
Sample type genomic
 
Channel 1
Source name orr_weaver_salivary_2 extraction2_array1 channel_1
Organism Drosophila melanogaster
Characteristics strain: Oregon-R Orr-Weaver
tissue: larval salivary gland
genotype: TBA
sex: Unknown
Growth protocol Salivary gland collection protocol; Growth Allow Modencode-OrR (MO) to lay embryos for 2-3 days, at 25C. Approximately three days later, collect (live) wandering 3rd instar larvae in Grace’s unsupplemented media. Dissection Carefully dissect out paired salivary glands from each larvae (Dumont #55 tweezers, INOX metallic alloy). Be sure to remove all contaminating fat body tissue. For CGH analysis, dissect approximately 150 paired salivary glands.
Extracted molecule genomic DNA
Extraction protocol DNA isolation WI protocol; For all tissues Protocol adapted from Qiagen DNeasy Blood and Tissue Handbook (July 2006). Using the DNeasy Blood and Tissue Kit: 1. Spin down samples and resuspend in 180uL ATL buffer. 2. Add 20uL proteinase K. Incubate at 56C until tissues are lysed (typically overnight for salivary gland tissues, four hours for other cell types). Vortex occasionally. 3. Vortex for 15s. Add 200uL buffer AL. Vortex again, and then add 200uL 100% EtOH. Vortex again to mix. 4. Pipet mixture into supplied DNeasy Mini Spin column placed in provided 2mL collective tube. Centrifuge for 1min at >6000g (14000rpm in a tabletop centrifuge is fine). 5. Place column in a fresh collection tube. Add 500uL buffer AW1, centrifuge for 1min at >6000g. Discard collection tube. 6. Transfer spin column to a fresh microcentrifuge tube. Pipet 50uL buffer AE onto column membrane. Incubate at room temperature for 1min, and elute by centrifuging for 1min at >6,000g.
Label Cy5
Label protocol Labeling WI protocol; For All Tissue Types Protocol Adapted from Agilent Technologies’ “Oligonucleotide Array-Based CGH for Genomic DNA Analysis” Protocol Handbook (Version 2.0, August 2005). The BioPrime Array CGH Genomic Labeling System (18095-011, Invitrogen) was used to prepare samples for CGH arrays. Cye-dyes were purchased from Amersham (25nmol, PA53022/PA55022). Label 3ug of genomic DNA (recovered from 150 larval salivary glands or 16C follicle cell nuclei from 300 pairs of ovaries). 50uL of embryos yields about 7-9ug of DNA. 1. Add 20uL of the 2.5x Random Primers solution to your previously prepared salivary gland gDNA. Transfer tube to a heat block at 95C and incubate for 5 minutes. Afterwards, immediately incubate sample on ice for an additional 5 minutes. 2. Prepare the labeling reaction mix on ice in the order indicated. 10x dUTP mix 5uL Cy3 or Cy5 –dUTP 3uL Exo-Klenow 1uL Mix this labeling reaction mix with your salivary gland gDNA (incubating on ice). 3. Transfer sample to a water bath at 37C for 2 hours. 4. Remove sample from water bath and add 5uL of Stop Buffer. 5. Concentrate labeled DNA with a microcon YM-30 filter according to manufacturer’s instructions for a final volume of 150uL, for each tube. 6. Quantitate total amount of incorporated dye in each sample using a Nanodrop. Combine up to 200 picomoles of labeled sample into a single tube, for a maximum volume of 150uL. Save the rest for technical replicates.
 
Channel 2
Source name orr_weaver_salivary_2 extraction2_array1 channel_2
Organism Drosophila melanogaster
Characteristics strain: Oregon-R Orr-Weaver
developmental stage: Embryo 0-4h
genotype: TBA
sex: Unknown
Growth protocol Embryo collection protocol; Using newly eclosed females fattened on wet yeast paste, collect embryos for up to four hours on apple juice plates. To ensure that females have not been holding embryos, precollect embryos for one hour. Wash embryos off the four-hour collection dish with PBS; dechorionate in 50% bleach for 3 minutes, and collect dechorionated embryos with a fine nylon mesh screen.
Extracted molecule genomic DNA
Extraction protocol DNA isolation WI protocol; For all tissues Protocol adapted from Qiagen DNeasy Blood and Tissue Handbook (July 2006). Using the DNeasy Blood and Tissue Kit: 1. Spin down samples and resuspend in 180uL ATL buffer. 2. Add 20uL proteinase K. Incubate at 56C until tissues are lysed (typically overnight for salivary gland tissues, four hours for other cell types). Vortex occasionally. 3. Vortex for 15s. Add 200uL buffer AL. Vortex again, and then add 200uL 100% EtOH. Vortex again to mix. 4. Pipet mixture into supplied DNeasy Mini Spin column placed in provided 2mL collective tube. Centrifuge for 1min at >6000g (14000rpm in a tabletop centrifuge is fine). 5. Place column in a fresh collection tube. Add 500uL buffer AW1, centrifuge for 1min at >6000g. Discard collection tube. 6. Transfer spin column to a fresh microcentrifuge tube. Pipet 50uL buffer AE onto column membrane. Incubate at room temperature for 1min, and elute by centrifuging for 1min at >6,000g.
Label Cy3
Label protocol Labeling WI protocol; For All Tissue Types Protocol Adapted from Agilent Technologies’ “Oligonucleotide Array-Based CGH for Genomic DNA Analysis” Protocol Handbook (Version 2.0, August 2005). The BioPrime Array CGH Genomic Labeling System (18095-011, Invitrogen) was used to prepare samples for CGH arrays. Cye-dyes were purchased from Amersham (25nmol, PA53022/PA55022). Label 3ug of genomic DNA (recovered from 150 larval salivary glands or 16C follicle cell nuclei from 300 pairs of ovaries). 50uL of embryos yields about 7-9ug of DNA. 1. Add 20uL of the 2.5x Random Primers solution to your previously prepared salivary gland gDNA. Transfer tube to a heat block at 95C and incubate for 5 minutes. Afterwards, immediately incubate sample on ice for an additional 5 minutes. 2. Prepare the labeling reaction mix on ice in the order indicated. 10x dUTP mix 5uL Cy3 or Cy5 –dUTP 3uL Exo-Klenow 1uL Mix this labeling reaction mix with your salivary gland gDNA (incubating on ice). 3. Transfer sample to a water bath at 37C for 2 hours. 4. Remove sample from water bath and add 5uL of Stop Buffer. 5. Concentrate labeled DNA with a microcon YM-30 filter according to manufacturer’s instructions for a final volume of 150uL, for each tube. 6. Quantitate total amount of incorporated dye in each sample using a Nanodrop. Combine up to 200 picomoles of labeled sample into a single tube, for a maximum volume of 150uL. Save the rest for technical replicates.
 
 
Hybridization protocol Agilent hybridization protocol; Setting up hybridization 1.Thaw samples on ice, in the dark 2.Set up hyb reaction: (Oligo aCGH/ChOP-on-Chip Hyb kit; Agilent) sample 25ul Ambion dH2O 175ul 10X Blocking Solution 50ul 2X Hybridization buffer 250ul 3.Inc 95C, 5 minutes 4.Spin down briefly and keep at 37C while getting slide ready 5.Place gasket with agilent side facing up on metal chamber 6.Pipet 490ul of hyb reaction on gasket 7.Lay down microarray on hyb reaction with Agilent side down. 8.Assemble metal chamber 9.inc. 16-20 hours in rotating hyb oven at 65C Washing microarrays 10.Dissamble arrays in tip box lid containing Buffer 1 (Agilent) 11.Wash slide in buffer 1 with stirring, 5 minutes 12.Wash slide in buffer 2 with stirring, 5 minutes 13.Wash slide in acetonitrile, 30 seconds 14.Wash slide in buffer 3, 30 seconds 15.Store in light proof holder and scan
Scan protocol Array scanning protocol; Scanning per Agilent recommendations.
Description N/A
Data processing Normalization and peak calling protocol; Agilent data files are converted to a nimblegen like format for processing by MA2C. pMA2C -- pyhton MA2C (http://liulab.dfci.harvard.edu/MA2C/MA2C.htm) Parameters are passed as a tag file which we generate in Array Scanning protocol. Processed data are obtained using following parameters: tag file is cgh/salivary/salivary_188.tag genome version is r5
 
Submission date Jul 23, 2009
Last update date Jul 29, 2009
Contact name DCC modENCODE
E-mail(s) help@modencode.org
Phone 416-673-8579
Organization name Ontario Institute for Cancer Research
Lab modENCODE DCC
Street address MaRS Centre, South Tower, 101 College Street, Suite 800
City Toronto
State/province Ontario
ZIP/Postal code M5G 0A3
Country Canada
 
Platform ID GPL7788
Series (1)
GSE17289 CGH Salivary Gland - OregonR

Supplementary file Size Download File type/resource
GSM432738_083006_251476110026_100100_CGH-v4_91.txt.gz 50.8 Mb (ftp)(http) TXT
GSM432738_Orr-Weaver_251476110026_532.txt.gz 933.5 Kb (ftp)(http) TXT
GSM432738_Orr-Weaver_251476110026_635.txt.gz 934.9 Kb (ftp)(http) TXT
Processed data are available on Series record

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