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Sample GSM2671860 Query DataSets for GSM2671860
Status Public on Mar 10, 2020
Title Myb mutant RNA polymerase Chip 2 Biological Replicate 1
Sample type genomic
 
Channel 1
Source name Input - Sonicated genomic DNA
Organism Drosophila melanogaster
Characteristics genotype: Myb^MH107: "mutation induced by P-element mobilization by Delta2-3 transposase"
phenotype: Lethal-early pupal stage
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 Cy3
Label protocol Labeling was performed following their standard protocol. See www.agilent.com.
 
Channel 2
Source name Immunoprecipitated RNA polymerase
Organism Drosophila melanogaster
Characteristics genotype: Myb^MH107: "mutation induced by P-element mobilization by Delta2-3 transposase"
phenotype: Lethal-early pupal stage
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
Label protocol Labeling was performed following their standard protocol. See www.agilent.com.
 
 
Hybridization protocol Hybridization was performed following their standard protocol. See www.agilent.com.
Scan protocol Scanning was performed following their standard protocol. See www.agilent.com.
Description Sample 1
Myb mutant imaginal wing discs of 3rd instar larvae. Chip two of biological replicate one
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)
 
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
Lab 260 EMRB
Street address 500 Newton Road
City Iowa City
State/province IA
ZIP/Postal code 52242
Country USA
 
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.

Supplementary file Size Download File type/resource
GSM2671860_myb_1_RNA_pol_chip_2_256490110014_1_S001_ChIP_1105_Oct12.txt.gz 96.4 Mb (ftp)(http) TXT
Processed data are available on Series record

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