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Sample GSM1192169 Query DataSets for GSM1192169
Status Public on Jul 22, 2013
Title Dichaete DamID vs control Dam rep1
Sample type genomic
 
Channel 1
Source name Dichaete DamID
Organism Drosophila melanogaster
Characteristics developmental stage: 0-12h embryos
genotype/variation: Flies contain a UAS-Dichaete-Dam fusion construct
tissue: Whole embryos
Treatment protocol Embryos were dechorionated, followed by homogenisation and DNA extraction
Growth protocol Embryos collected on agar plates with yeast at 25˚C
Extracted molecule genomic DNA
Extraction protocol DNA was extracted from embryos using a Qiagen Dneasy Blood & Tissue kit. It then underwent a series of digestions, followed by a PCR amplification, as described in the Vogel et al. 2007 DamID protocol.
Label Cy5
Label protocol To label take up to 1 µg double stranded DNA and make up to a total volume of 25 µl with DEPC-water. Add 20 µl 2.5x Random Primer Reaction Buffer (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 100 °C for 5 minutes, snap cool on ice. Add 1 µl 10 X low-C dNTP mix (5mM dATP,dGTP,dTTP, 2 mM dCTP), 2 µl 1 mM Cy3 or Cy5 dCTP (GE Healthcare) and 1 µl 40U/µl Klenow (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 37°C for 2 to 3 hours. Stop the reaction by adding 5 µl Stop Buffer (Invitrogen – BioPrime DNA Labelling Kit). Resuspend the resin in the AutoSeq G-50 column (GE Healthcare) by vortexing gently. Loosen the cap a quarter turn and snap off the bottom closure. Place the column in a collection tube. Pre-spin column at 5,000 rpm for 1 minute to remove the buffer. Remove the top cap and place column in a new 1.5 ml tube. Pipette half of the sample onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Spin for 1 minute at 5,000 rpm. Discard the column. Place a second column into the same 1.5 ml microfuge tube and then add the second half of the sample. Spin for 1 minute and 5,000 rmp. Reduce volume of probe to between 2 to 5 µl by placing in a speed vac with medium heat. Add 2 µl of 10 mg / ml sonicated salmon sperm DNA (Invitrogen).
 
Channel 2
Source name DamID control
Organism Drosophila melanogaster
Characteristics developmental stage: 0-12h embryos
genotype/variation: Flies contain a UAS-Dam construct
tissue: Whole embryos
Treatment protocol Embryos were dechorionated, followed by homogenisation and DNA extraction
Growth protocol Embryos collected on agar plates with yeast at 25˚C
Extracted molecule genomic DNA
Extraction protocol DNA was extracted from embryos using a Qiagen Dneasy Blood & Tissue kit. It then underwent a series of digestions, followed by a PCR amplification, as described in the Vogel et al. 2007 DamID protocol.
Label Cy3
Label protocol To label take up to 1 µg double stranded DNA and make up to a total volume of 25 µl with DEPC-water. Add 20 µl 2.5x Random Primer Reaction Buffer (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 100 °C for 5 minutes, snap cool on ice. Add 1 µl 10 X low-C dNTP mix (5mM dATP,dGTP,dTTP, 2 mM dCTP), 2 µl 1 mM Cy3 or Cy5 dCTP (GE Healthcare) and 1 µl 40U/µl Klenow (Invitrogen – BioPrime DNA Labelling Kit). Incubate at 37°C for 2 to 3 hours. Stop the reaction by adding 5 µl Stop Buffer (Invitrogen – BioPrime DNA Labelling Kit). Resuspend the resin in the AutoSeq G-50 column (GE Healthcare) by vortexing gently. Loosen the cap a quarter turn and snap off the bottom closure. Place the column in a collection tube. Pre-spin column at 5,000 rpm for 1 minute to remove the buffer. Remove the top cap and place column in a new 1.5 ml tube. Pipette half of the sample onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Spin for 1 minute at 5,000 rpm. Discard the column. Place a second column into the same 1.5 ml microfuge tube and then add the second half of the sample. Spin for 1 minute and 5,000 rmp. Reduce volume of probe to between 2 to 5 µl by placing in a speed vac with medium heat. Add 2 µl of 10 mg / ml sonicated salmon sperm DNA (Invitrogen).
 
 
Hybridization protocol Combined DNA samples were loaded onto Nimblegen D.mel ChIP 2.1M tiling arrays (GEO platform GPL15057), hybridised overnight at 42˚C in a NimbleGen hyb machine, then washed and scanned the following day.
Scan protocol Arrays are scanned at 5 µm resolution with a GenePix 4000B (Axon) dual laser scanner at 100% Power, with Lines to average = 1 and Focus position = 0 at their respective optimal PMT gains. Signal for Cy3 and Cy5 channels are balanced using the histogram view in the GenePix software. Images are saved as Single-image TIFF.
Description Dichaete 0-12h whole embryo DamID
Data processing NimbleScan was used for spot-finding. Scanned arrays were quantile normalised separately for samples and controls. The resulting ratios are log2 ratio of sample/control.
Peak finding was performed using RINGO to identify binding intervals at different False Discovery Rates.
 
Submission date Jul 18, 2013
Last update date Jul 22, 2013
Contact name Steve Russell
E-mail(s) [email protected]
Organization name University of Cambridge
Department Genetics
Street address Tennis Court Road
City Cambridge
ZIP/Postal code CB2 3EH
Country United Kingdom
 
Platform ID GPL15057
Series (2)
GSE49013 DamID experiment looking at Dichaete-bound regions in 2-7h old Drosophila embryos
GSE49095 The role of Dichaete in transcriptional regulation during Drosophila embryonic development

Data table header descriptions
ID_REF
VALUE quantile normalized log2 ratio (sample/control)

Data table
ID_REF VALUE
CHR02LFS000005131 3.438
CHR02LFS000005176 2.337
CHR02LFS000005236 2.951
CHR02LFS000005281 4.271
CHR02LFS000005401 2.85
CHR02LFS000005461 3.032
CHR02LFS000005501 3.041
CHR02LFS000005571 3.003
CHR02LFS000005611 3.213
CHR02LFS000005681 3.063
CHR02LFS000005736 2.824
CHR02LFS000005781 2.918
CHR02LFS000005831 2.703
CHR02LFS000005901 1.803
CHR02LFS000005941 1.623
CHR02LFS000006011 1.95
CHR02LFS000006051 2.18
CHR02LFS000006106 1.872
CHR02LFS000006171 1.752
CHR02LFS000006231 1.6

Total number of rows: 2157868

Table truncated, full table size 50662 Kbytes.




Supplementary file Size Download File type/resource
GSM1192169_539733_D1_532.pair.gz 33.5 Mb (ftp)(http) PAIR
GSM1192169_539733_D1_635.pair.gz 33.3 Mb (ftp)(http) PAIR
Processed data included within Sample table
Processed data are available on Series record

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