GSM2527016: mock_1 (2); Drosophila melanogaster; OTHER
Crosslinking was performed by adding formaldehyde directly to the media at final concentration of 1% and incubating for 10 min at RT. Reactions were quenched by adding glycine to a final concentration of 0.125 M and incubating for 5 min at RT. Reactions were incubated on ice 5 min followed by centrifugation at 1200 xg  at 4°C for 5 min. Cells were then washed with 5 mL of cold PBS and centrifuged at 1200 xg at 4°C for 5 min. Lysis was  performed by incubating cells in 1 mL of Lysis buffer (10 mM NaCl, 0.2% NP-40, 10 mM Tris pH8, 1 Mini Complete tablet (Roche) per 10 mL lysis buffer) at 37°C for 20 min. Samples were then centrifuged at 4200 xg at 4°C for 5 min, and the lysis step was repeated once. 800 μL of Lysis buffer was added, and samples were incubated at 37°C for 20 min. Samples were then centrifuged at 4200 xg at 4°C for 5 min, and resuspended in 1 mL Lysis buffer and incubated another 20 min at 37°C. Pellets from cell samples were then washed with 0.8 mL of digestion buffer [(0.2% NP-40, 1X NEBuffer 3 (NEB)] and centrifuged at 4200 xg at 4°C for 5 min. Nuclei were resuspended in 1.6 mL of digestion buffer with SDS added to a final concentration of 0.1% and incubated at 65°C for 30 min. Triton X-100 was added to a final concentration of 1% and samples incubated at 37°C for 15 min. A 40 μL aliquot of the sample was taken and used as the undigested control. The remaining sample was digested with 1600 U of HindIII (NEB) at 37°C overnight. Samples were incubated 20 min at 65°C to inactivate HindIII. A 40 μL aliquot of the sample was taken here and used as the digested control. The remaining sample was then diluted to 4 mL with ligation buffer [final concentrations were 1% Triton X-100, 1X T4 DNA Ligase Reaction Buffer (NEB)] and incubated at 37°C for 30 min. After the addition of 4800 U of T4 DNA Ligase (NEB), each sample was incubated at 16°C overnight. Proteinase K was added to all samples including the controls. Samples were then incubated at 65°C overnight to reverse crosslinking. After de-crosslinking, samples were combined with 1 vol of phenol/chloroform/isoamyl alcohol (25:24:1), vortexed 15 s, and centrifuged for 4 min at 10,000 xg. The top layer was transferred to a new tube, and the procedure was repeated using 1 vol chloroform. The top layer was collected and subsequently diluted with 1 vol of water. The sample was combined with 0.1 vol of 3M NaOAc pH 5.2 and 2.5 vol of 100% ethanol, supplemented with 2 μL of Glycoblue (Ambion). After incubating 1 h at -80°C, samples were centrifuged 20 min at 4°C at 10,000 xg. Pellets were washed with 75% ethanol and centrifuged 5 min at 4°C at 10,000 xg. Pellets were air dried at RT prior to resuspension in 350 μL water.  After resuspending the final pellet with 350 μl of dH2O, 300 μL of the 3C library was digested with 5 μL of DpnII (50 U, NEB) overnight at 37°C. After the enzyme was inactivated, digested chromatin was re-ligated using 20 μL of ligase (100 U, NEB) in a total volume of 8 mL ligation reaction. Finally purification of the library was carried out by adding 1 vol of phenol/chloroform/isoamyl alcohol (25:24:1), vortexed 15 s, and centrifuged for 4 min at 10,000 xg. The top layer was transferred to a new tube, and the procedure was repeated using 1 vol chloroform. The top layer was collected and precipitated with 0.1 vol of 3M NaOAc, pH 5.2 and 2.5 vol of 100% ethanol supplemented with 7 μL of Glycoblue. Inverse PCR was carried out using primers containing a 6 nucleotide barcode and one universal forward primer to produce Illumina-4C-seq libraries. PCR amplification was carried out using Phusion High Fidelity DNA Polymerase (NEB) with 60-70 ng of DNA and 1 μL of forward primer (10mM) and 1 μL of reverse primer (10mM). Amplification program: 95°C for 3 min, 30 cycles of 98°C for 20 s, 50°C for 1 min and 72°C for 2 min; final elongation step was 72°C for 5 min. After pooling together 3 PCR reactions, samples were purified using Agencourt AMPure XP beads (Beckman Coulter). 4C-seq
GEO Accession
GSM2527016