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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 18, 2010 |
| Title |
SOH10C3_24_vs_SOH10C5_23_247382-2 extraction2_array1 |
| Sample type |
RNA |
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| Channel 1 |
| Source name |
SOH10C3_24_vs_SOH10C5_23_247382-2 extraction2_array1 channel_1
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| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2(official name : N2 genotype : wild type genotype : DR subclone of DB original (Tc1 pattern I) )
developmental stage: Young Adult
genotype: wild type
Sex: Hermaphrodite
|
| Growth protocol |
Large cultures of transgenic worm strains were grown on enriched plates and embryos were isolated from gravid adult hermaphrodites by treatment with alkaline hypochlorite. The eggshells were removed by chitinase digestion, yielding embryonic blastomeres.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
C. elegans embryos are lysed in Trizol, extracted with CHCl3, and total cellular RNA is precipitated with isopropanol. Total RNA was used as a template to synthesize oligo(dT)-primed first-strand cDNA.
|
| Label |
Cy5 dye
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| Label protocol |
This is a modification of the NimbleGen protocol for ChIP-chip labeling (http://www.nimblegen.com/). It yields adequate amounts of end-labeled material without PCR amplification starting with ≥ 0.2 ug fragmented DNA.
1) In a 0.5 ml PCR tube, mix up to 20 uL ds DNA with 20 uL of either Cy3- or Cy5-labeled 9-mers in random primer buffer (Cy3 and Cy5 9-mer Wobble from TriLink are mixed according to the NimbleGen protocol, aliquoted and stored frozen). Use 0.2-3 ug input DNA, the more the better, and add water to 20 uL. Protect the dye-labeled material from direct light, especially Cy5, throughout the procedure.
2) Heat at 98oC x 10 min. Quick chill in ice-water (this is important).
3) While the samples are denaturing, make a master mix with Klenow exo- (NEB 50,000u/ml) and dNTPs. For example, for 10 samples mix 42 uL water, 10.5 uL Klenow, 52.5 uL 10mM dNTPs (aliquoted and stored frozen). While samples are still in the ice-water bath and chilled, add 10 uL master mix to each tube with 10 or more strokes of the Pipetteman tip.
4) Following a brief spin, transfer tubes to a 37oC cycler. Incubate overnight (16 hr).
5) Prepare 1.5 ml tubes with NaCl+EDTA (equivalent of 5 uL 0.5 M EDTA, 5.7 uL 5 M NaCl, which can be added together). Remove the tubes from the cycler and transfer contents from the cycler to these 1.5 ml tubes. Add 60 uL 2-propanol (the volume is important). Let sit in dark 10' at room temperature.
6) Spin at 13.2k rpm for 10 min at room temperature. Carefully decant the supernatant from the deeply colored pellet with a P200 Pipetteman. Add 500 uL ice-cold 80% ethanol and spin briefly in the cold. Carefully pour or pull off the ethanol - large pellets tend to dislodge. Quick spin and pull off the excess ethanol. Let air dry (~10 min at room temperature).
7) Add 20 uL water to dissolve. Note that the deepness of the red (Cy3) or blue (Cy5) is a good measure of the yield. For samples in which there was little starting material, and/or the DNA was small, there will not be enough for an array, and the color will be faint. For samples with deep color, add an additional 30 uL water. Measure OD260 and calculate yield. Store at 4oC for brief periods or at -20oC longer term in dark.
8) For samples with less than 30 ug/array, do another round of labeling, using 20 uL of the labeled DNA from the first round prep. The second round is much more efficient than the first round and consistently yields ≥ 60 ug.
9) Check sizes on a 1.5% gel without ethidium. Cy3 fluoresces well enough to see 1 ug, but Cy5 is much fainter and might not be visible. DNA should be about the same average size as the starting material. Follow with ethidium staining: DNA typically smears up towards the top of the gel, but this material is evidently low in molar amount of label and does not appear to cause a problem.
10) For each 2.1 M array, mix 30 ug Cy3-labeled DNA with 30 ug Cy5-labeled DNA and speed-vac dry on low or medium. If Cy3 is input DNA then Cy5 is pull-down DNA and vice versa.
11) Reduce volume by speedvac on low to 12.3 uL, or lyophilize samples for hybridization to NimbleGen arrays using standard NimbleGen hybridization protocol.
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| Channel 2 |
| Source name |
SOH10C3_24_vs_SOH10C5_23_247382-2_532 extraction4_array1 channel_2
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2(official name : N2 genotype : wild type genotype : DR subclone of DB original (Tc1 pattern I) )
developmental stage: Young Adult
genotype: wild type
Sex: Hermaphrodite
|
| Growth protocol |
Large cultures of transgenic worm strains were grown on enriched plates and embryos were isolated from gravid adult hermaphrodites by treatment with alkaline hypochlorite. The eggshells were removed by chitinase digestion, yielding embryonic blastomeres.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
C. elegans embryos are lysed in Trizol, extracted with CHCl3, and total cellular RNA is precipitated with isopropanol. Total RNA was used as a template to synthesize oligo(dT)-primed first-strand cDNA.
|
| Label |
Cy3 dye
|
| Label protocol |
This is a modification of the NimbleGen protocol for ChIP-chip labeling (http://www.nimblegen.com/). It yields adequate amounts of end-labeled material without PCR amplification starting with ≥ 0.2 ug fragmented DNA.
1) In a 0.5 ml PCR tube, mix up to 20 uL ds DNA with 20 uL of either Cy3- or Cy5-labeled 9-mers in random primer buffer (Cy3 and Cy5 9-mer Wobble from TriLink are mixed according to the NimbleGen protocol, aliquoted and stored frozen). Use 0.2-3 ug input DNA, the more the better, and add water to 20 uL. Protect the dye-labeled material from direct light, especially Cy5, throughout the procedure.
2) Heat at 98oC x 10 min. Quick chill in ice-water (this is important).
3) While the samples are denaturing, make a master mix with Klenow exo- (NEB 50,000u/ml) and dNTPs. For example, for 10 samples mix 42 uL water, 10.5 uL Klenow, 52.5 uL 10mM dNTPs (aliquoted and stored frozen). While samples are still in the ice-water bath and chilled, add 10 uL master mix to each tube with 10 or more strokes of the Pipetteman tip.
4) Following a brief spin, transfer tubes to a 37oC cycler. Incubate overnight (16 hr).
5) Prepare 1.5 ml tubes with NaCl+EDTA (equivalent of 5 uL 0.5 M EDTA, 5.7 uL 5 M NaCl, which can be added together). Remove the tubes from the cycler and transfer contents from the cycler to these 1.5 ml tubes. Add 60 uL 2-propanol (the volume is important). Let sit in dark 10' at room temperature.
6) Spin at 13.2k rpm for 10 min at room temperature. Carefully decant the supernatant from the deeply colored pellet with a P200 Pipetteman. Add 500 uL ice-cold 80% ethanol and spin briefly in the cold. Carefully pour or pull off the ethanol - large pellets tend to dislodge. Quick spin and pull off the excess ethanol. Let air dry (~10 min at room temperature).
7) Add 20 uL water to dissolve. Note that the deepness of the red (Cy3) or blue (Cy5) is a good measure of the yield. For samples in which there was little starting material, and/or the DNA was small, there will not be enough for an array, and the color will be faint. For samples with deep color, add an additional 30 uL water. Measure OD260 and calculate yield. Store at 4oC for brief periods or at -20oC longer term in dark.
8) For samples with less than 30 ug/array, do another round of labeling, using 20 uL of the labeled DNA from the first round prep. The second round is much more efficient than the first round and consistently yields ≥ 60 ug.
9) Check sizes on a 1.5% gel without ethidium. Cy3 fluoresces well enough to see 1 ug, but Cy5 is much fainter and might not be visible. DNA should be about the same average size as the starting material. Follow with ethidium staining: DNA typically smears up towards the top of the gel, but this material is evidently low in molar amount of label and does not appear to cause a problem.
10) For each 2.1 M array, mix 30 ug Cy3-labeled DNA with 30 ug Cy5-labeled DNA and speed-vac dry on low or medium. If Cy3 is input DNA then Cy5 is pull-down DNA and vice versa.
11) Reduce volume by speedvac on low to 12.3 uL, or lyophilize samples for hybridization to NimbleGen arrays using standard NimbleGen hybridization protocol.
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| |
| Hybridization protocol |
This protocol is copied from Chapter 4, Hybridization & Washing, in NimbleGen Arrays User's Guide ChIP-chip Analysis (Version 3.1, 27 May 2008), which describes the NimbleGen protocol for sample hybridization and washing.
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| Scan protocol |
Two-Color Array Scanning is copied from Chapter 5, Two-Color Array Scanning, in NimbleGen Arrays User's Guide ChIP-chip Analysis (Version 3.1, 27 May 2008), which describes the protocol for scanning two-color NimbleGen arrays with a GenePix 4000B Scanner and associated software
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| Data processing |
NimbleGen Scaling protocol. The log2-ratio ratio of intensity from sample/reference channel was scaled to center the ratio data around zero by subtracting the bi-weight mean for the log2-ratio values for all features on the array from each log2-ratio value. This was performed using NimbleScan software according to the NimbleScan v2.4 User's Guide and chapter 6 of the NimbleGen Arrays User's Guide ChIP-chip Analysis (version 3.1, 27 May 2008) at Roche NimbleGen. Processed data are obtained using following parameters: genome version is WS190.
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| Submission date |
Nov 17, 2010 |
| Last update date |
Apr 11, 2012 |
| Contact name |
DCC modENCODE |
| E-mail(s) |
[email protected]
|
| 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
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
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| Platform ID |
GPL7098 |
| Series (1) |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM624894_NimbHX_SOH10C3_24_vs_SOH10C5_23_247382-2_532.pair.gz |
33.7 Mb |
(ftp)(http) |
PAIR |
| GSM624894_NimbHX_SOH10C3_24_vs_SOH10C5_23_247382-2_635.pair.gz |
34.4 Mb |
(ftp)(http) |
PAIR |
| GSM624894_SOH10C3_24_vs_SOH10C5_23_247382-2_cy3_cy5_WS190.bedgraph.gz |
17.0 Mb |
(ftp)(http) |
BEDGRAPH |
| Processed data provided as supplementary file |
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