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| Status |
Public on Mar 10, 2020 |
| Title |
YW control RNA polymerase Chip 2 Biological Replicate 2 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
Input - Sonicated genomic DNA
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| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: Yellow white (yw^67)
phenotype: Yellow body and white eyes
gender: Male
developmental stage: 3rd instar larvae
tissue: Imaginal wing discs
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| Treatment protocol |
3rd instar larvae were dissected in 1X PBS (pH 7.4)
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| Growth protocol |
Flies were mantined at 25°C in malt-based media (Archon Scientific).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The tissue was fixed with 1.8% formaldehyde and incubated for 15 mins at room temperature on a rotating wheel. Crosslinking was stopped by adding glycine to a final concentration of 125mM in 0.1% PBS-Triton and incubated for 5 mins at room temperature. Samples were washed twice with lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 0.5% N-laurylsarcosine, and Roche Complete Protease Inhibitor Cocktail), snap-frozen in liquid nitrogen and stored at -80°C. Chromatin for each immunoprecipitation experiment was prepared from a total of 1000 wing imaginal discs and sonicated (30 pulses of 15 sec ON/ 15 sec OFF, high energy setting) in a Bioruptor (Diagenode) resulting in an average DNA fragment size of 400 bp. Samples were pre-cleared by adding 20 µl of Dynabeads® (Invitrogen) Protein A equilibrated in IP buffer (16.7 mM Tris-HCl, 167 mM NaCl, 1.2 Mm EDTA, 1.1% Triton, 0.1% SDS + 0.5% BSA) for 2 hrs at 4°C. 1% of the sample was saved as input. Immunoprecipitation was performed at 4°C overnight. The immunocomplexes were recovered by adding 20 µl of Dynabeads® Protein A (Invitrogen) (previously equilibrated) for 3 hrs at 4°C and washed with the following buffers: 1X for 10 mins with 1 ml of RIPA (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate); 5X for 10 mins with 1 ml of RIPA + 0.5M NaCl; 1X for 10 mins with 1 ml of LiCl buffer (250 mM LiCl, 10 mM Tris-HCl, pH 8, 1mM EDTA, 5% NP-40, 0.5% Sodium-deoxycholate ); and 2X for 10 mins with TE. DNA was released from the beads by adding elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) and incubating the samples at 65°C for 15 mins with occasional vortexing. TE was added to the eluted sample and input following by incubation in the presence of RNase A was to a final concentration of 0.2 mg/mL and incubated at 37°C for 1 hr. Proteinase K was added to a final concentration of 20 mg/mL and incubated at at 37°C overnight. The samples were then moved to 65°C and incubated for 6hrs to reverse the crosslinks. Finally, DNA was phenol/chloroform extracted and ethanol precipitated. DNA from the ChIP experiments were differentially labeled with Cy3- and Cy5-coupled random nanomers (Agilent), hybridized onto a custom Drosophila melanogaster tiled array
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| Label |
Cy3
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| Label protocol |
Labeling was performed following their standard protocol. See www.agilent.com.
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| Channel 2 |
| Source name |
Immunoprecipitated RNA polymerase
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: Yellow white (yw^67)
phenotype: Yellow body and white eyes
gender: Male
developmental stage: 3rd instar larvae
tissue: Imaginal wing discs
|
| Treatment protocol |
3rd instar larvae were dissected in 1X PBS (pH 7.4)
|
| Growth protocol |
Flies were mantined at 25°C in malt-based media (Archon Scientific).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The tissue was fixed with 1.8% formaldehyde and incubated for 15 mins at room temperature on a rotating wheel. Crosslinking was stopped by adding glycine to a final concentration of 125mM in 0.1% PBS-Triton and incubated for 5 mins at room temperature. Samples were washed twice with lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 0.5% N-laurylsarcosine, and Roche Complete Protease Inhibitor Cocktail), snap-frozen in liquid nitrogen and stored at -80°C. Chromatin for each immunoprecipitation experiment was prepared from a total of 1000 wing imaginal discs and sonicated (30 pulses of 15 sec ON/ 15 sec OFF, high energy setting) in a Bioruptor (Diagenode) resulting in an average DNA fragment size of 400 bp. Samples were pre-cleared by adding 20 µl of Dynabeads® (Invitrogen) Protein A equilibrated in IP buffer (16.7 mM Tris-HCl, 167 mM NaCl, 1.2 Mm EDTA, 1.1% Triton, 0.1% SDS + 0.5% BSA) for 2 hrs at 4°C. 1% of the sample was saved as input. Immunoprecipitation was performed at 4°C overnight. The immunocomplexes were recovered by adding 20 µl of Dynabeads® Protein A (Invitrogen) (previously equilibrated) for 3 hrs at 4°C and washed with the following buffers: 1X for 10 mins with 1 ml of RIPA (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate); 5X for 10 mins with 1 ml of RIPA + 0.5M NaCl; 1X for 10 mins with 1 ml of LiCl buffer (250 mM LiCl, 10 mM Tris-HCl, pH 8, 1mM EDTA, 5% NP-40, 0.5% Sodium-deoxycholate ); and 2X for 10 mins with TE. DNA was released from the beads by adding elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) and incubating the samples at 65°C for 15 mins with occasional vortexing. TE was added to the eluted sample and input following by incubation in the presence of RNase A was to a final concentration of 0.2 mg/mL and incubated at 37°C for 1 hr. Proteinase K was added to a final concentration of 20 mg/mL and incubated at at 37°C overnight. The samples were then moved to 65°C and incubated for 6hrs to reverse the crosslinks. Finally, DNA was phenol/chloroform extracted and ethanol precipitated. DNA from the ChIP experiments were differentially labeled with Cy3- and Cy5-coupled random nanomers (Agilent), hybridized onto a custom Drosophila melanogaster tiled array
|
| Label |
Cy5
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| Label protocol |
Labeling was performed following their standard protocol. See www.agilent.com.
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| |
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| |
| Hybridization protocol |
Hybridization was performed following their standard protocol. See www.agilent.com.
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| Scan protocol |
Scanning was performed following their standard protocol. See www.agilent.com.
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| Description |
Sample 4
Control imaginal wing discs of 3rd instar larvae. Chip two of biological replicate two
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| Data processing |
Raw data was extracted utilizing Agilent feature extraction V9.5.1. Data was normalized by applying blank subtraction, intra array (dye-bias) median and white error model normalization.
Blank substracted, intra-array (dye-bias), median normalized log2 ratio (ChIP/Input)
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| Submission date |
Jun 16, 2017 |
| Last update date |
Mar 11, 2020 |
| Contact name |
David H Price |
| E-mail(s) |
david-price@uiowa.edu
|
| Organization name |
University of Iowa
|
| Department |
Biochemistry and Molecular Biology
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| Lab |
260 EMRB
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| Street address |
500 Newton Road
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| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
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| Platform ID |
GPL23598 |
| Series (2) |
| GSE100138 |
Genome-wide analysis of RNA polymerase in Drosophila melangoaster Myb mutant wing discs of 3rd instar larvae. |
| GSE100143 |
Drosophila melangoaster Myb mutant wing discs of 3rd instar larvae. |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM2671866_yw_2_RNA_pol_chip_2_256490110013_2_S001_ChIP_1105_Oct12.txt.gz |
98.4 Mb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
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