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Status |
Public on Jul 22, 2013 |
Title |
Dichaete DamID vs control Dam rep1 |
Sample type |
genomic |
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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).
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|
|
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).
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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.
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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 |
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