Comment[ArrayExpressAccession] E-GEOD-47338 MAGE-TAB Version 1.1 Public Release Date 2014-04-23 Investigation Title Analysis of SoxNeuro gene expression changes, genome-wide binding, and comparison of SoxNeuro and Dichaete binding profiles in wild type and mutant embryos Comment[Submitted Name] Analysis of SoxNeuro gene expression changes, genome-wide binding, and comparison of SoxNeuro and Dichaete binding profiles in wild type and mutant embryos Experiment Description This SuperSeries is composed of the SubSeries listed below. Refer to individual Series 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 Ferrero Ferrero Fischer Russell Person First Name Enrico Enrico Bettina Steven Person Email ef300@cam.ac.uk Person Affiliation University of Cambridge Person Address Genetics, University of Cambridge, Downing Street, Cambridge, United Kingdom Person Roles submitter Protocol Name P-GSE47338-2 P-GSE47338-1 P-GSE47338-8 P-GSE47338-10 P-GSE47338-5 P-GSE47338-6 P-GSE47338-11 P-GSE47338-3 P-GSE47338-9 P-GSE47338-13 P-GSE47338-4 P-GSE47338-12 P-GSE47338-7 Protocol Description Images are processed with NimbleScan software (v.2.6, Roche-Nimblegen) as per manufacturer instructions. Raw data files (*.pair) are created for each channel. Perform Nimblescan loess spatial correction and quantile normalise the raw data. Supplemetary processed data *quant-all.sgr file description: the first field is the chromosome ("chr"), the second field is the starting nucleotide position of the oligo on the array ("nt"), and the third is a score representing the log2 ratio of sample intensity over control intensity for that oligo, aka M-value ("score"). "quant-all" refers to the fact that scores/M-values/log2 ratios have been quantile normalised. ID_REF = VALUE = Quantile normalised log2 ratio (sample/control) Images are processed and spot quantified by the Dapple software (v.0.88, http://www.cs.wustl.edu/~jbuhler/research/dapple/). Raw data are then loaded into limma (Bioconductor) and normalised with the vsn package using the limma package (R. 2.15 and Bioconductor v. 2.11). The resulting ratios are log2 ratio of sample/control (where SoxN heterozygous embryos are the controls, while SoxN homozygous embryos are considered the samples). ID_REF = VALUE = VSN normalised log2 ratio (sample/control) To 3 to 5 M-5g of total RNA, add 1 M-5l 500 ng/ M-5l of Oligo(dT)23 anchored primer (Sigma) and adjust volume to 5 M-5l with DEPC- water. Incubate at 65 M-0C for 10 min, then snap cool on ice. Add 4 M-5l 5x First Strand Buffer (Invitrogen), 2 M-5l 0.1M DTT (Invitrogen), 1 M-5l 10 mM dNTP mix, 0.25 M-5l 40 U/M-5l RNasin (Promega) and 0.75 M-5l 200 U/M-5l Superscript III (Invitrogen). Incubate at 46 M-0C for 2 hours, then at 65 M-0C for 15 minutes, snap cool on ice. For second strand synthesis: Add 7.5 M-5l Second Strand Buffer (Invitrogen), 0.75 M-5l 10 mM dNTP mix, 2 M-5l 10 U/M-5l DNA Polymerase I (Invitrogen), 0.1 M-5l 5 U/M-5l RnaseH (Promega), 0.5 M-5l 10 U/M-5l E. coli Ligase (NEB) adjust volume to 40 M-5l with DEPC-water. Incubate at 16 M-0C for 2 hours, then purification using QIAquick PCR Purification Kit. In PCR tubes take 1 M-5g double stranded DNA, add 40 diluted Cy3 or Cy5-Random Nonamers (Nimblegen Dual Colour Kit) and make up to a volume of 80 M-5l with Nuclease-free Water (Nimblegen Dual Colour Kit). Heat-denature samples in a thermocycler at 98M-0C for 10 minutes. Quickchill on ice for 10 minutes. Add 10 M-5l 10mM dNTP Mix (Nimblegen Dual Colour Kit), 8 Nuclease-free Water (Nimblegen Dual Colour Kit) and 2 M-5l 50U/M-NM-5M-bM-^@M-^Y exo-) (Nimblegen Dual Colour Kit), mix gently by pipetting. Incubate at 37M-0C for 3 hours in thermocycler. Stop the reaction by adding 21.5 M-5l Stop Solution (Nimblegen Dual Colour Kit). Transfer samples to 1.5 ml tubes and add 110 M-5l Isopropanol, mix and incubate for 10 minutes at room temperature in the dark. Centrifuge at 13,000rpm for 10 minutes and discard supernatant. Add 500 M-5l ice-cold 80% ethanol and centrifuge at 13,000rpm for 2 minutes. Discard supernatant and dry pellet for 5 minutes in the dark. Resuspend labeled DNA in 50 M-5l Nuclease-free Water (Nimblegen) and measure the concentration on the Nanodrop. Combine 34 M-5g of sample with 34 M-5g control and dry samples in a speed vac with medium heat. In PCR tubes take 1 M-5g double stranded DNA, add 40 diluted Cy3 or Cy5-Random Nonamers (Nimblegen Dual Colour Kit) and make up to a volume of 80 M-5l with Nuclease-free Water (Nimblegen Dual Colour Kit). Heat-denature samples in a thermocycler at 98M-0C for 10 minutes. Quickchill on ice for 10 minutes. Add 10 M-5l 10mM dNTP Mix (Nimblegen Dual Colour Kit), 8 Nuclease-free Water (Nimblegen Dual Colour Kit) and 2 M-5l 50U/M-NM-5M-bM-^@M-^Y exo-) (Nimblegen Dual Colour Kit), mix gently by pipetting. Incubate at 37M-0C for 3 hours in thermocycler. Stop the reaction by adding 21.5 M-5l Stop Solution (Nimblegen Dual Colour Kit). Transfer samples to 1.5 ml tubes and add 110 M-5l Isopropanol, mix and incubate for 10 minutes at room temperature in the dark. Centrifuge at 13,000rpm for 10 minutes and discard supernatant. Add 500 M-5l ice-cold 80% ethanol and centrifuge at 13,000rpm for 2 minutes. Discard supernatant and dry pellet for 5 minutes in the dark. Resuspend labeled DNA in 50 M-5l Nuclease-free Water (Nimblegen) and measure the concentration on the Nanodrop. Combine 34 M-5g of sample with 34 M-5g control and dry samples in a speed vac with medium heat. Adjust RNA volume to 3 M-5L with DEPC-water and add 1 M-5L 3'-SMART CDS Primer IIA (10 M-5M) and 1 M-5L SMART IIA chimeric oligo (10 M-5M). Incubate at 70 M-0C for 5 minutes, then snap cool on ice. Add 2 M-5l 5x First Strand Buffer (Invitrogen), 0.5 M-5L 0.1M DTT (Invitrogen), 0.5 M-5L 10 mM dNTP mix and 1 M-5l 200 U/M-5L Superscript III (Invitrogen). Incubate at 46 M-0C for 1.5 hours, then cool on ice (store samples at 4 M-0C or -20 M-0C). To determine the optimal number of cycles required for generating products in exponential phase, 2 M-5L of the PCR products from a parallel run are visualised at every second cycle from cycle 18 to 26 on 1% agarose gel using one reaction of each sample type. To 5 M-5L of the first-strand reaction (include negative and positive controls) add 5 M-5L 10x Advantage II PCR buffer (Clontech), 1 M-5L 10 mM dNTP mix, 2 M-5L 20 M-5M 5' PCR Primer II, 1 M-5L 50x Advantage II Polymerase Mix (Clontech) and adjust volume to 50 M-5L with DEPC-water. Run PCR program: step 1: 95 M-0C - 1 min, step 2: 95 M-0C - 5 sec, step 3: 65 M-0C - 5 sec, step 4: 68 M-0C - 6 min, cycle from step 2 for 27 more times. Run PCR aliquots on 1% agarose gel and select cycle number below saturation. Then perform amplification using the optimal number of XX cycles on the remaining 5 M-5L of the first-strand reaction. Purify PCR products using QIAquick PCR Purification Kit. 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). SMART II A chimeric primer: 5'-AAG-CAG-TGG-TAT-CAA-CGC-AGA-GTA-CGC-888-3' (8 = riboG). 3' SMART CDS Primer IIA: 5'-AAG-CAG-TGG-TAT-CAA-CGC-AGA-GTA-CTT-TTT-TTT-TTT-TTT-TTT-TTT-TTT-TTT-TTT-VN-3' (V = G+A+C, N = A+C+G+T). 5' PCR Primer II: 5'-AAG-CAG-TGG-TAT-CAA-CGC-AGA-GT-3'. In PCR tubes take 1 M-5g double stranded DNA, add 40 diluted Cy3 or Cy5-Random Nonamers (Nimblegen Dual Colour Kit) and make up to a volume of 80 M-5l with Nuclease-free Water (Nimblegen Dual Colour Kit). Heat-denature samples in a thermocycler at 98M-0C for 10 minutes. Quickchill on ice for 10 minutes. Add 10 M-5l 10mM dNTP Mix (Nimblegen Dual Colour Kit), 8 Nuclease-free Water (Nimblegen Dual Colour Kit) and 2 M-5l 50U/M-NM-5M-bM-^@M-^Y exo-) (Nimblegen Dual Colour Kit), mix gently by pipetting. Incubate at 37M-0C for 3 hours in thermocycler. Stop the reaction by adding 21.5 M-5l Stop Solution (Nimblegen Dual Colour Kit). Transfer samples to 1.5 ml tubes and add 110 M-5l Isopropanol, mix and incubate for 10 minutes at room temperature in the dark. Centrifuge at 13,000rpm for 10 minutes and discard supernatant. Add 500 M-5l ice-cold 80% ethanol and centrifuge at 13,000rpm for 2 minutes. Discard supernatant and dry pellet for 5 minutes in the dark. Resuspend labeled DNA in 50 M-5l Nuclease-free Water (Nimblegen) and measure the concentration on the Nanodrop. Combine 34 M-5g of sample with 34 M-5g control and dry samples in a speed vac with medium heat. Add 140 M-5l of Ocimum hybridisation buffer to the labelled mixture and heat at 100 M-0C for 2 minutes on a hot-block. Centrifuge for 3 minutes at 13,000 rpm. Load 140 M-5l of the labelled sample to the microarray, and hybridise for 16 hours at 51 M-0C. Perform post-hybridisation washes as per PowerMatrix slides protocol (FMB). Pre-heat wash solution 1 (0.2 x SSC; 0.2% SDS) and wash solution 2 (0.2 x SSC) to 55 M-0C using a water bath. Incubate slides in a slide staining dish with pre-heated wash solution 1 on an orbital shaker at 50 rpm for 20 minutes. Transfer the slides to a staining trough containing pre-heated wash solution 2. Gently dip the slides up and down for 1 minute in wash solution 2. Repeat step in fresh wash solution 2 twice. Rinse the slides 3 times with fresh deionized water at room temperature (dip the rack in MilliQ water for 3 seconds). Transfer the slides to a clean microscope slide box with tissue at the base and centrifuge at 1000 rpm for 5 minutes. The slides are now ready to be scanned. Resuspend samples in 12.3 M-5l water. For each hybridisation, combine 29.5M-5l 2X Hybridization Buffer, 11.8M-5l Hybridization Component A and 1.2M-5l Alignment Oligo (NimbleGen Hybridization Kit) and add 31.7M-5l to each hybridisation sample. Incubate samples at 95M-0C for 5 minutes, 42M-0C for 5 minutes and load 41 M-5l onto the array. The hybridisation is performed to NimbleGen D. melanogaster ChIP-chip 2.1M Whole-Genome Tiling Arrays in the Nimbelegen hybridisation station at 42M-0C (mix mode B) for 16 hours. Perform post hybridisation washes according to the NimbleGen Wash Buffer Kit instructions. Grow flies at 25M-0C; collect and age embryos on grape agar juice plates supplemented with yeast paste, at 25M-0C Extract genomic DNA from dechorionated embryos with the Qiagen DNeasy blood and tissue kit and precipitate with ethanol. Digest DNA overnight at 37M-0C with DpnI to cut methylated GATC sequences and ligate the fragments to a double-stranded adapter oligonucleotide. Digest ligation products with DpnII to remove unmethylated GATC sequences and PCR amplify. See Vogel et al., 2007 for a detailed protocol. Crosslink dechorionated embryos with formaldehyde, isolate and lyse nuclei to extract protein-DNA complexes. Incubate chromatin overnight poth protein A agarose beads / salmon sperm DNA and anti-M-NM-2gal or anti-SoxN antibodies. After extensive washes, reverse crosslinking by incubating at 65M-0C for 6 hours and purify DNA trough phenol-chloroform extraction. Amplify DNA with two rounds of ligation-mediated PCR. See Sandmann et al., 2007 for a detailed protocol. Homogenise tissue in 300 M-5l TRIzol (Invitrogen) in a 1.5 ml tube using an RNAase-free polypropylene pellet pestle. Centrifuge at 13,000 rpm in a microcentriuge for 10 minutes 4 M-0C to pellet debris and transfer supernatant to a fresh 1.5 ml tube. Add 0.2 volumes chloroform, vortex for 60 seconds. Centrifuge at 13,000 rpm for 15 minutes. Remove upper phase to a new RNase-free tube, being careful not to touch the interface. Add 0.4 M-5l GeneElute Linear Polyacrylamide (Sigma) (25 M-5g / M-5l stock diluted 1:10 in DEPC MilliQ water) and 0.8 volumes of isopropanol, invert and then incubate for 1 hour at -20M-0C. Pellet the RNA by centrifugation at 13,000 rpm for 30 minutes. Discard the supernatant and wash the RNA pellet with 500 M-5l 70% ethanol/DEPC MilliQ water. Air-dry the pellet. Resuspend in an appropriate volume of DEPC MilliQ water, e.g. 5 M-5l. Verify quality of RNA on a 0.8 % agarose gel stained with SYBR-gold (Molecular Probes). 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. 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 selected by the Auto-PMT function (0.005% saturation tolerance). Images are saved as Single-image TIFF. Protocol Type normalization data transformation protocol normalization data transformation protocol labelling protocol labelling protocol labelling protocol hybridization protocol hybridization protocol growth protocol nucleic acid extraction protocol nucleic acid extraction protocol nucleic acid extraction protocol array scanning protocol array scanning protocol Experimental Factor Name DEVELOPMENTAL STAGE STRAIN Experimental Factor Type developmental stage strain Comment[SecondaryAccession] GSE47338 Comment[GEOReleaseDate] 2014-04-23 Comment[ArrayExpressSubmissionDate] 2013-05-23 Comment[GEOLastUpdateDate] 2014-04-24 Comment[AEExperimentType] transcription profiling by array Comment[AEExperimentType] ChIP-chip by tiling array SDRF File E-GEOD-47338.sdrf.txt