Comment[ArrayExpressAccession]	E-GEOD-50768
MAGE-TAB Version	1.1							
Public Release Date	2013-09-15
Investigation Title	Mapping the inter- and intra-chromosomal interactions of specific insulator binding sites with circular chromosome conformation capture (4C) assay (Gondor et al., 2008 Nature Protocol) [NimbleGen Array data]							
Comment[Submitted Name]	Mapping the inter- and intra-chromosomal interactions of specific insulator binding sites with circular chromosome conformation capture (4C) assay (Gondor et al., 2008 Nature Protocol) [NimbleGen Array data]
Experiment Description	Drosophila Insulator proteins mediate long-range chromosomal interactions. ChIP-seq revealed that binding of insulator proteins to some specific DNA sites was regulated by poly(ADP-ribosyl)ation in S2 cells. Three insulator sites regulated by poly(ADP-ribosyl)ation were used as baits to map their distant interacting sites using 4C assay in control S2 cells. Mapping the chromosomal interactions of three specific insulator binding sites with 4C assay in control S2 cells.							
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	Ong	Corces	Ong					
Person First Name	Chintong	Victor	Chin-Tong					
Person Email	ongchintong@yahoo.com							
Person Affiliation	Emory University							
Person Address	Biology, Emory University, 1510 Clifton Road NE, Atlanta, GA, USA							
Person Roles	submitter							
Protocol Name	P-GSE50768-1	P-GSE50768-5	P-GSE50768-7	P-GSE50768-2	P-GSE50768-6	P-GSE50768-3	P-GSE50768-4	P-GSE50768-8
Protocol Description	Data processing was performed by the FSU-NimbleGen Microarray Facility. Validation experiments were carried out only on intra-chromosomal interactions between distant insulator binding sites. Briefly, interacting 4C sites (identified by the microarray) that contained CP190 and/or dCTCF (from ChIP-seq) were determined using Galaxy. These sites were then ranked by their signal strength (defined by peak height and width). At least 50 interacting 4C sites from each bait and verified were subjected to PCR using site-specific primers in multiple 3C libraries. The map illustrated in Figure 5 showed only 4C interactions that are verified by site-specific primers in multiple 3C libraries. ID_REF =  VALUE = normalized ratio Cy5/Cy3	Labeling was performed by the FSU-NimbleGen Microarray Facility according to the NimbleGen protocol.	Hybridization was performed by the FSU-NimbleGen Microarray Facility according to the NimbleGen protocol.	Libraries prepared with 4C protocol (Gondor et. al., Nature Protocol, 2008.) using DpnII. DNA were ligated and subjected to 2 separate rounds on inverse PCR using specific primers pair.	Genomic DNA cut with DpnII	Cells were cultured in Schneider's medium with 10% HI FBS.	4C libraries were prepared from S2-DRSC cells using DpnII enzymes according to Gondor et. al., Nature Protocol 2008 and Ong et. al., Cell 2013. The detailed protocol is described in the following steps: 1. 25 million S2-DRSC cells were centrifuged for 5 min at 1000 rpm. The cell pellet was resuspended with 10 ml of fresh medium. Cells were fixed by adding 278 M-NM-<l of 37% (vol/vol) formaldehyde and incubated at RT for 10 min with gentle shaking. 2. The cross-linking was quenched by adding 1.142 ml of 1.25 M glycine to cells and incubating for 5 min at RT, followed by 15 min on ice. Fixed cells were then centrifuged for 5 min at 1000 rpm (Eppendorf 5810 R). After removing supernatant, the cell pellet was froze in liquid nitrogen and stored at M-bM-^HM-^R80 M-0C for later use. 3. Frozen cell pellet was thawed on ice and resuspended in cold lysis buffer (10 mM Tris pH 8, 10 mM NaCl and 0.2% NP-40 supplemented with Roche cOmplete Mini EDTA-free protease inhibitor cocktail). 1 ml of lysis buffer was used for 50 million cells. 4. The lysate was incubated on ice for at least 15 min, with gentle mixing every 5 min. After incubation, the cells were homogenized on ice with two rounds of 15 strokes in a Dounce homogenizer (pestle A). Cell lysate was then transferred to a 1.5 ml microfuge tube and centrifuged for 5 min at 3000 rpm at RT. 5. The pellet (from 50 million cells) obtained was resuspended in 500 M-NM-<l of 1M-CM-^W DpnII restriction buffer. After centrifugation for 5 min at 3000 rpm at RT, the pellet collected was washed again with 500 M-NM-<l of 1M-CM-^W DpnII restriction buffer. 6. After centrifugation, the pellet was resuspended in 500 M-NM-<l of 1M-CM-^W DpnII restriction buffer and divided equally into 50 M-NM-<l aliquot. Each aliquot, which contains nuclei from approximately 5 million cells, can be frozen in liqiud nitrogen and stored at -80 M-0C for several months. Frozen pellet of nuclei was thawed on ice before use. 7. The thawed aliquot was mixed thoroughly with 337 M-NM-<l of 1 M-CM-^W restriction buffer, followed by 38 M-NM-<l of 1% SDS. The mixture was then incubated for 1 hr at 37 M-0C with gentle shaking. 8. The activity of SDS was sequestered by adding 44 M-NM-<l of 10% Triton X-100. 30 M-5l of the mixture was kept as undigested fraction. 9. 400 U of DpnII per tube (5 million cells) was mixed thoroughly with the nuclei and the mixture was incubated overnight at 37 M-0C with gentle shaking. 10. 200 U of DpnII was further added to each tube and incubated for additional 4 hr at 37 M-0C. 30 M-5l of the restriction mixture was kept as digested fraction. 11. The DpnII was inactivated by adding 86 M-NM-<l of 10% SDS and incubating at 65 M-0C for 30 min. 12. Each reaction (~575 M-NM-<l) was then transferred individually to a 15-ml falcon tube. 13. The ligation reaction was then assembled by adding the following reagents in the order of: 745 M-NM-<l of 10% Triton X-100, 745 M-NM-<l of 10 x T4 DNA ligase buffer, 80 M-NM-<l of BSA (10mg/ml), 80 M-NM-<l of 100 mM ATP, 5960 M-NM-<l of water and 10 M-NM-<l of T4 ligase (400U/ M-NM-<l). The reaction was mixed thoroughly and incubated at 16 M-0C for 4 hr, followed by at RT for 1 hr to facilitate circularization of DNA. 14. After ligation, 50 M-NM-<l of proteinase K (10mg/ml) was added and incubated overnight at 65 M-0C. 15. On the following day, 35 M-5g of RNase A (Sigma) was added and incubated at 37M-oM-^BM-0C for 30 min. This was followed by addition of 50 M-NM-<l of proteinase K (10mg/ml) and 2 hr of incubation at 65 M-0C. 16. Each ligation reaction was transferred into 50-ml conical tubes containing 8 ml of phenol. The mixture was vortex for 2 min and centrifuged for 30 min at 4000 rpm and 4 M-0C (Eppendorf 5810R). 17. The upper aqueous layer was then transferred to a fresh 50-ml conical tube and added with 8 ml phenol/chloroform. The mixture was vortex and centrifuged for 30 min at 3000 rpm and 4 M-0C. 18. The upper aqueous layer was transferred to a fresh 50-ml conical tube and added with 7 ml of nuclease-free water, 1.5 ml of 3 M NaAc (pH 5.2), 35 ml of ethanol and incubated at M-bM-^HM-^R80 M-0C for at least 30 min. The mixture was then centrifuged for 1 hr at 4000 rpm and 4 M-0C. 19. After discarding the supernatant, the pellet was dissolved in 500 M-NM-<l of 1 M-CM-^W TE buffer (pH 8) by incubating at 42 M-oM-^BM-0C for 10 min and then at RT for 10 min. The dissolved DNA was then transferred to a 1.5 ml microfuge tube. 20. After adding 500 M-NM-<l of phenol/chloroform, the mixture was vortex for 1 min and centrifuged for 10 min at 14,000 rpm in a microfuge at RT. 21. The upper aqueous layer was transferred to a new microfuge tube and added with 0.1 volumes of 3 M NaAc (pH 5.2) and 2 volumes of cold ethanol. The mixture was incubated at M-bM-^HM-^R80 M-0C for at least 30 min and centrifuged for 20 min at 16,000 rpm at 4 M-0C. 22. The supernatant was discarded and the white pellet was resuspended with 1 ml of 70% ethanol. The mixture was centrifuge for 5 min at 16,000 rpm at 4 M-0C. 23. Ethanol wash described in steps 23 was repeated for at least seven more times. 24. After the final ethanol wash, the pellet was air-dried and dissolved in 50 M-NM-<l of TE (pH 8). 25. Digestion efficiency was determined with the undigested and digested fractions using previously described method (Hagege et al., 2007). The DpnII digestion efficiency is between 70-95% depending on the DNA sites examined. 26. DNA concentration was measured with Qubit Fluorometer (Invitrogen). The yield of 3C library from 5 million cells was typically 40-50 ng/M-5l in 50 M-5l total volume. Generation of 4C libraries 27. The following method was adapted from (Gondor et al., 2008). Duplicate inverse PCRs for each cross-linked sample was set up with the composition of one reaction as follow: Water (final volume of 40 M-5l), 8 M-5l of 5x HF buffer, 2 M-5l each of forward and reverse primers (10 M-5M), 1.4 M-5l of dNTP (10 mM), 0.8 M-5l of MgCl2 (10 mM), 0.6 M-5l of DMSO, 0.6 M-5l of Phusion high-fidelity DNA polymerase (NEB) and 50 ng of 3C DNA. 28. The first inverse PCR was carried out with the following cycling parameters: (1) 30 sec 98 M-0C. (2) 4 cycles of 10 sec 98 M-0C, 40 min 65 M-0C, 5 min 72 M-0C. (3) 20 rounds of 7 sec 98 M-0C, 2 min 30 sec 58 M-0C, 5 min 72 M-0C. (4) 7 min 72 M-0C (5) forever 4 M-0C. 29. The inverse PCR product obtained was diluted 80 fold (i.e. 1 M-5l product with 79 M-5l TE buffer). The dilution concentration was determined empirically in which the linear phase of amplification was attained after 30 cycles of nested PCR (Figure S5B). 30. Three nested PCRs for each core inverse PCR product was set up with the composition of one reaction as follow: Water (final volume of 20 M-5l), 4 M-5l of 5x HF buffer, 1 M-5l each of forward and reverse nested primers (10 M-5M), 0.7 M-5l of dNTP (10 mM), 0.4 M-5l of MgCl2 (10 mM), 0.3 M-5l of DMSO, 0.3 M-5l of Phusion high-fidelity DNA polymerase (NEB) and 0.2 ng of diluted inverse PCR DNA. 31. The nested PCR was carried out with the following cycling parameters: (1) 30 sec 98 M-0C. (2) 30 rounds of 10 sec 98 M-0C, 30 sec 55 M-0C, 2 min 72 M-0C. (3) 10 min 72 M-0C (4) forever 4 M-0C. 32. Nested PCR products were pooled and purified with QIAquick PCR purification kit (QIAGEN). Pooled 4C samples and DpnII digest genomic DNA were hybridized to Drosophila melanogaster ChIP-chip 2.1M Whole-Genome Tiling Array (Roche) at the NimbleGen Microarray Facility at Florida State University. 33. To reduce technical/biological variations, the 4C-chip assay was represented by four independently cross-linked samples (four separate 3C libraries) undergoing the 4C protocol. In total, there are 4 x 2 x 3 = 24 nested PCR reactions.	Scanning was performed by the FSU-NimbleGen Microarray Facility according to the NimbleGen protocol.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	MOLECULE TYPE							
Experimental Factor Type	molecule type							
Comment[SecondaryAccession]	GSE50768							
Comment[GEOReleaseDate]	2013-09-15							
Comment[ArrayExpressSubmissionDate]	2013-09-11							
Comment[GEOLastUpdateDate]	2013-09-15							
Comment[AEExperimentType]	ChIP-chip by tiling array							
SDRF File	E-GEOD-50768.sdrf.txt