|
| Status |
Public on Mar 25, 2011 |
| Title |
MG_CGH extraction2_array2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
MG_CGH extraction2_array2 channel_1
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Oregon-R(official name : Oregon-R-modENCODE genotype : wild type )
tissue: midgut
developmental stage: L3 stage wandering larvae
genotype: wild type
|
| Growth protocol |
From a Drosophila culture, manually remove larvea of selected type.
Collection and dissection of biologicial tissues, to include:embryo 0-2hfollicle cellsnurse cellsadult fat bodysalivary gland
|
| 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.
|
| |
|
| Channel 2 |
| Source name |
MG_CGH extraction2_array2 channel_2
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Oregon-R(official name : Oregon-R-modENCODE genotype : wild type )
tissue: midgut
developmental stage: L3 stage wandering larvae
genotype: wild type
|
| Growth protocol |
From a Drosophila culture, manually remove larvea of selected type.
Collection and dissection of biologicial tissues, to include:embryo 0-2hfollicle cellsnurse cellsadult fat bodysalivary gland
|
| 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.
|
| |
|
| |
| 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 |
This scanning protocol outputs raw images of intensity values resulting from the hybridization of the DNA sample to the microarray slide. The raw tiffs are quantified and output in an Agilent text format. We then mangle the Agilent data into a pseudo nimblegen format.
|
| Data processing |
Normalization_and_peak_calling:DM:1 protocol. 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 http://macalpine-lab.duhs.duke.edu/files/modencode/Midgut_CGH/400K_Array/midgut_400K.tag
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| |
|
| Submission date |
Mar 25, 2011 |
| Last update date |
Apr 11, 2012 |
| 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 |
GPL11290 |
| Series (1) |
| GSE28179 |
Midgut - CGH Differential Replication |
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