GSM4314686: 4C_DM_HS+.rep1; Drosophila melanogaster; OTHER
DNA samples were prepared as described before (Dekker et al., 2002; Osborne et al., 2004; http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3978).  Cross-linking: To suspension containing about 34x106 S2 cells in 4 ml of the GIBCO medium formaldehyde solution was added to final concentration 1.5%. After mixing incubation of cell suspension was performed for 10 min at room temperature with mixing. The quenching with 2.75 ml of 2M glycin (final concentration 0.125M) was performed. After incubation at room temperature for 5 min the suspension was cooled for 15 min in ice bath and then cells were collected by centrifugation for 15 min at 3500 rpm at +2°C.  Lysis: The pellet of cells was resuspended at °C in 1ml of buffer containing 10 mM tris-HCl buffer, pH 8, 10 mM NaCl, 0.2% NP-40 and freshly added protease inhibitors – 0.1 mM PMSF and 1:500 protease inhibitor cocktail (Sigma). After incubation for 15 min cells were homogenized by passing through insulin syringe about 50 times into Eppendorf. Then nuclei were spin down by centrifugation for 5 min at 5000 rpm in Eppendorf centrifuge 5415 R at +2°C.  Digestion with EcoRI: The nuclei pellet was resuspended in 756 microl of solution containing 40 mM tris-HCl buffer, pH 7.4, 50 mM NaCl, 10 mM MgCl2 and 10 mM 2-mercaptoethanol. Then 20% SDS was added to final concentration 0.3% and incubation with shaking was performed for 1 h at +37°C. To sequester SDS 180 l of 10% Triton X-100 was added and the incubation for 1 h at +37°C was performed. Before digestion 1 l of BSA (5 mg/ml) was added with mixing. Then 50 l of EcoRI (10 u/l) was added and after mixing digestion was performed overnight at +37°C.   Ligation: To inactivate the restriction enzyme, 35 l of 20% SDS was added (final concentration 0.7%) and the probe was heated to 65°C for 30 min. Then the mixture was transferred into 15 ml Nunc tube and consequently 375 l of 20% Triton X-100 (to final concentration 1%), 750 l of 10x ligase buffer, 7,5 l of BSA (5 mg/ml), 80 l of 100 mM ATP, and 5241 l of milliQ water  were added and the final 7.5 ml solution was well mixed. Then 10 l of T4 DNA ligase (200 u/l) was added and after mixing incubation was performed for 5 h at +16°C and then for 30 min at room temperature. Usually during ligation DNA concentration was equal to 2 ng/l.    DNA purification: For isolation of DNA 50 l of proteinase K (10 mg/ml) was added (final concentration 50 g/ml) and after mixing incubation was performed at 55°C B1overnight. For RNA digestion 40l of RNase A (10 mg/ml) was added (final concentration 0.5 g/ml) and after mixing incubation was performed for 30 min at +37°C. After extensive extraction with phenol-chloroform extraction (three times with 7 ml each) DNA was precipitated by 2.5 vol. of ethanol after addition of 40 l 10 mg/ml glycogen and 175 l 4M NaCl. The final DNA pellet was washed twice with 70% ethanol and then dissolved in 0.1xTE.   Digestion with FaeI: About 15 g of DNA was digested in 250 microl solution with 75 u of FaeI overnight at +37°C. Then the enzyme was inactivated by heating at 65°C for 30 min. DNA was isolated after phenol-chloroform extraction, precipitated by ethanol and dissolved in 100 l of 0.1xTE.   Ligation: For circularization 15 microg of DNA was incubated in 8 ml of T4 DNA ligase buffer containing 400 u of T4 DNA ligase for 5 h at +16°C. DNA was isolated after phenol-chloroform extraction, precipitated by ethanol and dissolved in 50 microl of 0.1xTE. One  round of  PCR was used as described below. The following primers were used: 5' ACTGTTCACGATGAACTTGGAATT 3' and 5' TTCAGTTCACAATCCCAAGCATG 3'. PCR products were purified using QIAqiuck PCR purification kit kit (Quigen). The libraries were prepared using TruSeq RNA Sample Preparation Kit v2 (Illumina). The samples were sequenced using HiSeq 1500 (Illumina) following the manufacturer's protocols.
GEO Accession
GSM4314686