Comment[ArrayExpressAccession] E-GEOD-49013 MAGE-TAB Version 1.1 Public Release Date 2013-07-22 Investigation Title DamID experiment looking at Dichaete-bound regions in 2-7h old Drosophila embryos Comment[Submitted Name] DamID experiment looking at Dichaete-bound regions in 2-7h old Drosophila embryos Experiment Description Dichaete is a developmentally important transcription factor, known to be involved in basic biological processes including segmentation and nervous system development among others. The aim of this experiment was to gain further insight into the role of Dichaete during early embryogenesis, by finding out where in the genome it's binding using the DamID technique. 3 independent biological replicates. Embryos between 2-7 h old were collected. The sample embryos contained a Dichaete-Dam methylase fusion, while the control embryos had a Dam methylase only to simulate the background level of methylation not specific to transcription factor binding. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Russell Ferrero Aleksic Fischer Russell Person First Name Steve Enrico Jelena Bettina Steve Person Email sr120@hermes.cam.ac.uk Person Affiliation University of Cambridge Person Address Genetics, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom Person Roles submitter Protocol Name P-GSE49013-1 P-GSE49013-5 P-GSE49013-6 P-GSE49013-2 P-GSE49013-3 P-GSE49013-4 P-GSE49013-7 Protocol Description 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. ID_REF = VALUE = quantile normalized log2 ratio (sample/control) To label take up to 1 M-5g double stranded DNA and make up to a total volume of 25 M-5l with DEPC-water. Add 20 M-5l 2.5x Random Primer Reaction Buffer (Invitrogen M-bM-^@M-^S BioPrime DNA Labelling Kit). Incubate at 100 M-0C for 5 minutes, snap cool on ice. Add 1 M-5l 10 X low-C dNTP mix (5mM dATP,dGTP,dTTP, 2 mM dCTP), 2 M-5l 1 mM Cy3 or Cy5 dCTP (GE Healthcare) and 1 M-5l 40U/M-5l Klenow (Invitrogen M-bM-^@M-^S BioPrime DNA Labelling Kit). Incubate at 37M-0C for 2 to 3 hours. Stop the reaction by adding 5 M-5l Stop Buffer (Invitrogen M-bM-^@M-^S 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 M-5l by placing in a speed vac with medium heat. Add 2 M-5l of 10 mg / ml sonicated salmon sperm DNA (Invitrogen). Combined DNA samples were loaded onto Nimblegen D.mel ChIP 2.1M tiling arrays (GEO platform GPL15057), hybridised overnight at 42M-KM-^ZC in a NimbleGen hyb machine, then washed and scanned the following day. Embryos were dechorionated, followed by homogenisation and DNA extraction Embryos collected on agar plates with yeast at 25M-KM-^ZC 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. Arrays are scanned at 5 M-5m 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. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name GENOTYPE Experimental Factor Type genotype Comment[SecondaryAccession] GSE49013 Comment[GEOReleaseDate] 2013-07-22 Comment[ArrayExpressSubmissionDate] 2013-07-18 Comment[GEOLastUpdateDate] 2013-07-22 Comment[AEExperimentType] ChIP-chip by tiling array Comment[AdditionalFile:Data1] GSE49013_P40092_quant-sep_Ringo_rel5_hw300v5g200FDR1.bed Comment[AdditionalFile:Data2] GSE49013_P40092_quant-sep_Ringo_rel5_hw300v5g200FDR25.bed SDRF File E-GEOD-49013.sdrf.txt