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Sample GSM659163 Query DataSets for GSM659163
Status Public on Jan 21, 2011
Title UBX_Leg
Sample type genomic
 
Channel 1
Source name Leg (T3) imaginal disc ChIP; yw strain; Ubx antibody
Organism Drosophila melanogaster
Characteristics strain: yw
developmental stage: L3
tissue: T3 leg imaginal disc
chip antibody: anti-Ubx
chip antibody provider: Kevin White lab
Growth protocol Wandering 3rd instar larvae were manually removed from Drosophila culture. Larvae were inverted in PBS to expose imaginal discs. Imaginal discs dissected off after fixation.
Extracted molecule genomic DNA
Extraction protocol Third instar larvae were dissected in PBS and fixed for 20 minutes at room temperature in 1.8% formaldehyde, 50mM HEPES, 1mM EDTA, 0.5mM EGTA, 100mM NaCl. After quenching (0.125M Glycine, 1xPBS, 0.01% Triton) the fixed tissue was washed first in Buffer A (10mM HEPES, 10mM EDTA, 0.5mM EGTA, 0.25% Triton, 1mM PMSF), then in Buffer B (10mM HEPES, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.01% Triton, 1mM PMSF), for 10 minutes at 4C. Imaginal discs were then dissected off the cuticles in Buffer B. Discs were sonicated in Buffer C (10mM HEPES, 1mM EDTA, 0.5mM EGTA, 1mM PMSF, plus complete protease inhibitor cocktail (Roche)) on ice/EtOH. Soluble chromatin was transferred to new tubes after centrifugation. Fresh chromatin, precleared with protein G sepharose beads (Roche), was incubated overnight at 4C with antibody in RIPA buffer (140mM NaCl, 10mM HEPES, 1mM EDTA, 1% Glycerol, 1% Triton X-100, 0.1% DOC, 1mM PMSF). Antibody-chromatin complexes were pulled down with protein G sepharose beads for 3 hours at 4C. Bound beads were washed 4-times in RIPA buffer (as above plus 0.1mg/mL ssDNA) and once in TE. Chromatin was eluted twice in Elution Buffer (1% SDS, 0.1M NaHCO3). 20ug Proteinase K was added, and samples were incubated for 3 hours at 55C, then 65C overnight. DNA was purified via phenol-chloroform extraction and ethanol precipitation.
Label biotin
Label protocol ChIP and Input DNA amplified using Sigma Whole Genome Amplifcation kit (WGA4). After amplification, labeling proceeds as follows: Use the Bioprime DNA labeling Kit from Invitrogen, 20 ul of DNA (around 300ng/ul of PCR-purified DNA) 20 ul of 2.5X Random Primers solution. Boil at 95C for 5 min, return to ice. Add 5ul of 10X dnTP+U and 3ul of water, flick mix, spin. Add 2ul of Klenow. Incubate 2.5 hours at 37C, then add 5ul of stop buffer. Purify using PCR purification kit, elute in 30 ul of buffer. Quantify the amount of DNA using the Nanodrop spectrophotometer, and use 7.5 ug of DNA for susequent reaction. Use the Affymetrix double-stranded DNAlabeling kit to label DNA. Prepare labeling mix -- 10X cDNA fragmentation buffer UDG, 10 U/ul APE1, 100 U/ul Nuclease-free H2O up to 48ul. Incubate 1 hour at 37 C. Boil at 93C for 2 min, let stand on ice for at least 2 min. Label the fragmented double-stranded DNA with 5X TdT buffer (12ul) TdT (2ul) 5mM DNA labeling reagent (1ul). Add the 15ul Mix to the DNA samples. Incubate 60 min at 37C, 10 min at 70C and at least 2min on ice.
 
Channel 2
Source name Leg (T3) imaginal disc ChIP; yw strain [input DNA]
Organism Drosophila melanogaster
Characteristics strain: yw
developmental stage: L3
tissue: T3 leg imaginal disc
chip antibody: none
Growth protocol Wandering 3rd instar larvae were manually removed from Drosophila culture. Larvae were inverted in PBS to expose imaginal discs. Imaginal discs dissected off after fixation.
Extracted molecule genomic DNA
Extraction protocol Third instar larvae were dissected in PBS and fixed for 20 minutes at room temperature in 1.8% formaldehyde, 50mM HEPES, 1mM EDTA, 0.5mM EGTA, 100mM NaCl. After quenching (0.125M Glycine, 1xPBS, 0.01% Triton) the fixed tissue was washed first in Buffer A (10mM HEPES, 10mM EDTA, 0.5mM EGTA, 0.25% Triton, 1mM PMSF), then in Buffer B (10mM HEPES, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.01% Triton, 1mM PMSF), for 10 minutes at 4C. Imaginal discs were then dissected off the cuticles in Buffer B. Discs were sonicated in Buffer C (10mM HEPES, 1mM EDTA, 0.5mM EGTA, 1mM PMSF, plus complete protease inhibitor cocktail (Roche)) on ice/EtOH. Soluble chromatin was transferred to new tubes after centrifugation. Fresh chromatin, precleared with protein G sepharose beads (Roche), was incubated overnight at 4C with antibody in RIPA buffer (140mM NaCl, 10mM HEPES, 1mM EDTA, 1% Glycerol, 1% Triton X-100, 0.1% DOC, 1mM PMSF). Antibody-chromatin complexes were pulled down with protein G sepharose beads for 3 hours at 4C. Bound beads were washed 4-times in RIPA buffer (as above plus 0.1mg/mL ssDNA) and once in TE. Chromatin was eluted twice in Elution Buffer (1% SDS, 0.1M NaHCO3). 20ug Proteinase K was added, and samples were incubated for 3 hours at 55C, then 65C overnight. DNA was purified via phenol-chloroform extraction and ethanol precipitation.
Label biotin
Label protocol ChIP and Input DNA amplified using Sigma Whole Genome Amplifcation kit (WGA4). After amplification, labeling proceeds as follows: Use the Bioprime DNA labeling Kit from Invitrogen, 20 ul of DNA (around 300ng/ul of PCR-purified DNA) 20 ul of 2.5X Random Primers solution. Boil at 95C for 5 min, return to ice. Add 5ul of 10X dnTP+U and 3ul of water, flick mix, spin. Add 2ul of Klenow. Incubate 2.5 hours at 37C, then add 5ul of stop buffer. Purify using PCR purification kit, elute in 30 ul of buffer. Quantify the amount of DNA using the Nanodrop spectrophotometer, and use 7.5 ug of DNA for susequent reaction. Use the Affymetrix double-stranded DNAlabeling kit to label DNA. Prepare labeling mix -- 10X cDNA fragmentation buffer UDG, 10 U/ul APE1, 100 U/ul Nuclease-free H2O up to 48ul. Incubate 1 hour at 37 C. Boil at 93C for 2 min, let stand on ice for at least 2 min. Label the fragmented double-stranded DNA with 5X TdT buffer (12ul) TdT (2ul) 5mM DNA labeling reagent (1ul). Add the 15ul Mix to the DNA samples. Incubate 60 min at 37C, 10 min at 70C and at least 2min on ice.
 
 
Hybridization protocol Hybridization Cocktail (for 1 reaction): DNA (around 60 ul); Control Oligo B2 (3.3 ul); 2X Hybridization Buffer (110 ul); DMSO (15.4 ul); Water (31.3 ul), for a total of 220 ul volume per sample. Boil 5 min at 99 C Cool to room temperature. Hybridizations are done in the core facility as per the Affy protocol.
Scan protocol Arrays were scanned on an Affymetrix Scanner
Description Ubx leg disc ChIP, replicates 1-3
Data processing ChIP-chip data were generated using Affymetrix Drosophila tiling array 2.0 and are subjected to the analysis using TAS (Tiling Analysis Software, Affymetrix) and MAT (model based analysis of tiling arrays; Johnson et al, 2006). In MAT, all Control and IP samples corresponding to the same experiment are analyzed together. We specified the MAT parameters Bandwidth, MaxGap and Minprobe parameters to be 200, 100 and 10 respectively. We used non-redundant dm3 BPMAP file and self generated Repeat Library files available for download at http://liulab.dfci.harvard.edu/MAT/Download.htm. Peaks were called at 5% FDR.
Processed files descriptions: All .bar files were generated by Affymetrix TAS software. signal.bar files contain log2(IP/Input) fold changes for each probe. pvalue.bar files contain p-value for each probe. Ubx_Haltere_MAT-FDR5.bed, Ubx_Leg_MAT-FDR5.bed, Hth_Haltere_MAT-FDR5.bed, Hth_Leg_MAT-FDR5.bed contain peaks called by MAT software (Johnson et al, PNAS, 2006) at 5% FDR. Ubx_Haltere_TAS-Top5.bed, Ubx_Leg_TAS-Top5.bed, Hth_Haltere_TAS-Top5.bed, Hth_Leg_TAS-Top5.bed contain the top 5% of peaks, based on p-value, as calculated by TAS.
 
Submission date Jan 21, 2011
Last update date Feb 02, 2015
Contact name Matthew Slattery
Organization name University of Minnesota Medical School
Street address 1035 University Drive, SMed 219
City Duluth
State/province MINNESOTA
ZIP/Postal code 55812
Country USA
 
Platform ID GPL6629
Series (2)
GSE23537 modENCODE_White Lab: genome-wide ChIP-chip and ChIP-Seq data
GSE26793 Genome-wide binding of Ubx and Hth in 3rd thoracic imaginal discs
Relations
Named Annotation GSM659163_Ubx_Leg_MAT-FDR5.bed.gz
Named Annotation GSM659163_Ubx_Leg_TAS-Top5.bed.gz

Supplementary file Size Download File type/resource
GSM659163_Leg_Ubx-Input_A.CEL.gz 35.0 Mb (ftp)(http) CEL
GSM659163_Leg_Ubx-Input_B.CEL.gz 33.7 Mb (ftp)(http) CEL
GSM659163_Leg_Ubx-Input_C.CEL.gz 34.2 Mb (ftp)(http) CEL
GSM659163_Leg_anti-Ubx_A.CEL.gz 34.8 Mb (ftp)(http) CEL
GSM659163_Leg_anti-Ubx_B.CEL.gz 34.6 Mb (ftp)(http) CEL
GSM659163_Leg_anti-Ubx_C.CEL.gz 34.2 Mb (ftp)(http) CEL
GSM659163_UBX_Leg_TAS-pvalue.bar.gz 13.2 Mb (ftp)(http) BAR
GSM659163_UBX_Leg_TAS-signal.bar.gz 17.2 Mb (ftp)(http) BAR
GSM659163_Ubx_Leg_MAT-FDR5.bed.gz 36.7 Kb (ftp)(http) BED
GSM659163_Ubx_Leg_TAS-Top5.bed.gz 7.9 Kb (ftp)(http) BED
Processed data provided as supplementary file

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