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Sample GSM2671877 Query DataSets for GSM2671877
Status Public on Mar 10, 2020
Title Myb mutant H3K27me3 Chip 1 Biological Replicate 2
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 H3K27me3
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 2
Myb mutant imaginal wing discs of 3rd instar larvae. Chip one of biological replicate two
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 GPL23597
Series (2)
GSE100140 Genome-wide analysis of trimethylation of lysine 27 of histone 3 (H3K27me3) 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
GSM2671877_myb_2_H3K27_chip_1_256492810004_2_S001_ChIP_1105_Oct12.txt.gz 97.0 Mb (ftp)(http) TXT
Processed data are available on Series record

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