Comment[ArrayExpressAccession]	E-GEOD-45072
MAGE-TAB Version	1.1														
Public Release Date	2013-03-13
Investigation Title	NURF301_Q2602.BG3														
Comment[Submitted Name]	NURF301_Q2602.BG3
Experiment Description	modENCODE_submission_5063 This submission comes from a modENCODE project of Gary Karpen. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We aim to determine the locations of 125 chromosomal proteins across the Drosophila melanogaster genome. The proteins under study are involved in basic chromosomal functions such as DNA replication, gene expression, gene silencing, and inheritance. We will perform Chromatin ImmunoPrecipitation (ChIP) using genomic tiling arrays. We will initially assay localizations using chromatin from three cell lines and two embryonic stages, and will then extend the analysis of a subset of proteins to four additional animal tissues/stages 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-chip. BIOLOGICAL SOURCE: Cell Line: ML-DmBG3-c2; Tissue: CNS-derived cell-line; Developmental Stage: third instar larval stage; Genotype: y v f mal; Sex: Unknown;  NUMBER OF REPLICATES: 4;  EXPERIMENTAL FACTORS: Cell Line ML-DmBG3-c2; Antibody NURF301 Q2602 (target is NURF301)														
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	Karpen	Elgin	Gortchakov	Shanower	Tolstorukov	Kharchenko	Kuroda	Pirrotta	Park	Minoda	Riddle	Schwartz	Alekseyenko	Kennedy
Person First Name	DCC	Gary	Sarah	Andrey	Greg	Michael	Peter	Mitzi	Vincenzo	Peter	Aki	Nicole	Yuri	Art	Cameron
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-GSE45072-4	P-GSE45072-3	P-GSE45072-1	P-GSE45072-2	P-GSE45072-5										
Protocol Description	Standard Affymetrix Array Scanning Protocol was used	1. Prehybridize array for 1 hr in 200ml of 1xMES-Triton at 450C with 45 rpm rotation. 2. Incubate hybridization cocktail for 10min at 1000C then 10min at 450C. Spin at max speed for 3 min at RT. Transfer the supernatant to new tube. Spin for 3 more minutes use 200ml of resulted supernatant for hybridization. 3. Hybridize 18hr at 450C with 45 rpm rotation. 4. Use fluidics station EukGE-WS2v4 protocol (Affymetrix) for washing and staining.	1. Grow cell cultures in large T-flasks (175-225cm2) to a density of~5x106 cells/ml. Combine the content of 2-3 flasks in one. Immediately proceed with the following steps. 2. Add 37% formaldehyde (Sigma) to final concentration 1.8% directly to the culture incubate for 10min at 250C on a rotating or rocking platform. 3. Stop reaction by adding 1.25M glycine pH 7.0 to each culture to the final concentration of 125mM. Mix well, within next 5 min aliquot cell suspension into 50ml falcon tubes and accumulate them on ice. 4. Pellet fixed cells by spinning for 2? at 1500g, +40C. Use swinging bucket rotor. 5. Resuspend each pellet in 5ml of sterile 1xPBS, pool suspensions together. 6. Pellet the cells by spinning for 2? at 1500g, +40C. 7. Resuspend the cells in 10ml of ChIP wash A solution. Transfer suspension to a new 15ml Falcon tube. Wash fixed cells for 10min at +40C using rotating wheel. Pellet as above. 8. Thoroughly resuspend fixed cells in 10ml of ChIP wash B solution, wash for 10min at +40C. Use rotating wheel. After treatment with formaldehyde, Drosophila tissue culture cells were lysed with SDS and sonication (on ice) in the presence of protease inhibitors. The resulting chromatin was treated with non-ionic detergents at physiological concentrations of monovalent cations. Ribonucleic acid was removed by RNAse A, and the DNA was purified using SDS, proteinase K, and organic extraction, followed by ethanol precipitation. 1. Add 30ml of PAS (50% suspension in RIPA (-PMSF)) to 500ml of crosslinked chromatin. Incubate 1h at +40C. 2. Spin suspensions for 2min at top speed +40C. Transfer supernatants to new tubes. Add 5ml of 100mM PMSF solution in isopropanol to each 500ml aliquot of precleared chromatin. 3. Add appropriate amount of antibody to each reaction. Do not forget to set up no Ab control. Incubate for 15 hours at +40C on rotating weel. 4. Add 40ml of PAS (50% suspension in RIPA (-PMSF)), incubate 3h at +40C on rotating weel. 5. Wash the beads 5 times 10min each with 1ml of RIPA, then one time with 1ml of LiCl ChIP buffer and finally twice with 1ml of TE. To pellet the beads between washes spin samples for 20sec +40C at top speed. Do all the washes at +40C. 6. Resuspend the beads in 100ml TE add 1ml (final 50mg/ml) of RNAse A (10mg/ml) incubate 30min at +370C. 7. Add 7.5ml (final 0.5%) of 10% SDS and 3.8ml (final 0.5mg/ml) of Proteinase K (20mg/ml). Incubate overnight at +370C. 8. Transfer samples at +650C, incubate 6h. 9. Add 4.5ml of 5M NaCl (140mM final). Extract samples with 150ml of phenol-chloroform by vortexing for 30 sec, centrifuge for 10 min at RT, take 120ml of aqueous phase, back-extract organic phase with 150ml of TEN 140 (10mM Tris-HCl pH8.0; 1mM EDTA; 140mM NaCl). Take 150ml of aqueous phase. Combine aqueous phases (you will get 120ml + 150ml=270ml of solution). 10. Extract samples with 300ml of chloroform by vortexing for 30 sec, centrifuge for 5 min at RT. Transfer the upper aqueous phase into the new tube. Add 30ml of 3M NaAc pH 5.0 and 2ml of glycogen (5mg/ml) to aqueous phase. Precipitate DNA with 900ml of EtOH at -700C for 1h. 11. Spin for 10min, top speed at +40C. Wash the pellet in 300ml of 70%EtOH 12. Spin for 10min, top speed at +40C. If you plan to do qPCR analysis only dissolve the pellet in 150ml of pure H2O. If you plan to do both qPCR and microarray hybridization then first dilute DNA pellets in 12ml of pure H2O transfer 4ml of DNA solution to a new eppendorf tube and add 46ml of pure H2O. Use the latter for qPCR and the former for subsequent amplification and labeling. Store DNA solutions at -200C. 13. Prepare dilutions of DNA isolated from the original crosslinked chromatin (also called ?Input DNA? or simply ?Input?) following the chart below. Use the stock with concentration of 0.5% of input DNA per ml of solution (see: ?Isolation of ChIP Input DNA? protocol).	1. Prehybridize array for 1 hr in 200ml of 1xMES-Triton at 450C with 45 rpm rotation. 2. Incubate hybridization cocktail for 10min at 1000C then 10min at 450C. Spin at max speed for 3 min at RT. Transfer the supernatant to new tube. Spin for 3 more minutes use 200ml of resulted supernatant for hybridization. 3. Hybridize 18hr at 450C with 45 rpm rotation. 4. Use fluidics station EukGE-WS2v4 protocol (Affymetrix) for washing and staining.	M-value normalization protocol. The log-intensity ratio values (M-values) are calculated for all perfect match (PM) probes as log2(ChIP intensity) - log2(input intensity). The M values are then shifted so that the mean is equal to 0.										
Protocol Type	image_aquisition	hybridization	nucleic_acid_extraction	labeling	feature_extraction										
Comment[SecondaryAccession]	GSE45072														
Comment[GEOReleaseDate]	2013-03-13														
Comment[ArrayExpressSubmissionDate]	2013-03-12														
Comment[GEOLastUpdateDate]	2013-03-14														
Comment[AEExperimentType]	ChIP-chip by tiling array														
Comment[AdditionalFile:Data1]	GSE45072_repset.16023710.smoothedM.bedgraph														
SDRF File	E-GEOD-45072.sdrf.txt