Comment[ArrayExpressAccession]	E-GEOD-49776
MAGE-TAB Version	1.1			
Public Release Date	2013-08-11
Investigation Title	Su(H)_0-8_embryonic_ChIP-seq			
Comment[Submitted Name]	Su(H)_0-8_embryonic_ChIP-seq
Experiment Description	modENCODE_submission_5017 This submission comes from a modENCODE project of Kevin White. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: The White Lab is aiming to map the association of all the Transcription Factors (TF) on the genome of Drosophila melanogaster. One technique that we use for this purpose is chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) utilizing an Illumina next generation sequencing platform. The data generated by ChIP-seq experiments consist basically of a plot of signal intensity across the genome. The highest signals correspond to positions in the genome occupied by the tested TF. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: Y cn bw sp; Developmental Stage: Embryo 0-8; Genotype: y[1] oc[R3.2]; Gr22b[1] Gr22d[1] cn[1] CG33964[R4.2] bw[1] sp[1]; LysC[1] lab[R4.2] MstProx[1] GstD5[1] Rh6[1]; Sex: Unknown;  EXPERIMENTAL FACTORS: Developmental Stage Embryo 0-8; Strain Y cn bw sp; Antibody Su(H) (target is fly genes:Su(H))			
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	modENCODE	White	Ma	Slattery
Person First Name	DCC	Kevin	Lijia	Matthew
Person Email	help@modencode.org			
Person Affiliation	Ontario Institute for Cancer Research			
Person Phone	416-673-8579			
Person Address	Ontario Institute for Cancer Research, MaRS Centre, South Tower, 101 College Street, Suite 800, Toronto, Ontario, Canada			
Person Roles	submitter			
Protocol Name	P-GSE49776-1			
Protocol Description	1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder. 2. For a 8-16 hours egg laying, new plates are added after a 2 hours pre-egglaying at 12 pm and flies are allowed to lay eggs for 8 hours. 3. Embryos are collected at 7.45 am using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 8am in 1.8% formaldehyde at room temperature for 15 min. Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample. DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911). After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.			
Protocol Type	nucleic acid library construction protocol			
Comment[SecondaryAccession]	GSE49776			
Comment[GEOReleaseDate]	2013-08-11			
Comment[ArrayExpressSubmissionDate]	2013-08-11			
Comment[GEOLastUpdateDate]	2013-08-11			
Comment[AEExperimentType]	ChIP-seq			
Comment[AdditionalFile:Data1]	GSE49776_5017_peaks.GFF3			
SDRF File	E-GEOD-49776.sdrf.txt