GSM5463980: shCtrl_E2_4Cseq_GREB1; Homo sapiens; OTHER
4C-seq was performed following Brouwer et al. 2017 (16). Specifically, 8-10 x 10^6 cells from each experimental condition were resuspended in 10ml of culture media and 1ml of 16% methanol-free formaldehyde (Thermo Fisher Scientific 28908) was added to the cell suspension for a final concentration of 1.45% to crosslink the cells at room temperature for 10min. The crosslinking reaction was quenched with 575ml of 2.5M glycine (Sigma-Aldrich G8898). The cells were then centrifuged for 8min at 340 x g at 4°C before being lysed 10-15min on ice in 5ml of prechilled lysis buffer (10mM tris-HCl pH 8, 10mM NaCl, 0.2% NP-40) supplemented with protease inhibitors (Sigma-Aldrich S8830). Nuclei from the lysed cells were pelleted by centrifuging at 650 x g at 4°C for 5min, washed with 1x PBS, and aliquoted into 4-5 x 106 nuclei pellets before flash freezing and storing at -80°C. One aliquot of each experimental condition was resuspended in 0.5 ml of 1.2x buffer B (Thermo Fisher Scientific BB5) followed by incubation with 15ml of 10% SDS then with 50 ml of 20% Triton X-100, each for 1h at 37°C with gentle agitation at 900rpm. A 10ml aliquot was removed and stored at -20°C prior to chromatin digestion with 400U of CviQI (Thermo Fisher Scientific ER0211) overnight at 37°C and gentle agitation at 900rpm. A 10ml aliquot was removed prior to decrosslinking with 10ml of proteinase K (NEB P8107S) and 10mM tris-HCl pH 8 at 37°C for 1h in a final volume of 100ml. Digestion efficiency was determined by running 20ml of the pre- and post-digestion samples on a 0.6% agarose gel. If a smear was observed, CviQI was heat inactivated by incubating at 65°C for 20min. To finish generating the 3C library, the samples were transferred to a 50ml tube for ligation overnight at 16°C with 6.125ml of 1.15x T4 ligation buffer (NEB B0202S) and 100U of T4 DNA ligase (NEB M0202L). Next, samples were decrosslinked with 30ml of proteinase K at 65°C for at least 4h and treated with 30ml of RNase A (10mg/ml, Thermo Fisher Scientific AM9780) for 30min at 37°C. After cooling the samples down to room temperature, 7ml of phenol/chloroform/isoamyl alcohol (Thermo Fisher Scientific 15-593-049) was added and samples were shaken vigorously then centrifuged at 3200 x g for 15min. The upper aqueous layer was transferred to a new 50ml tube to which 7ml of water, 1.5ml of 3M sodium acetate (pH 5.4), and 35ml of 100% ethanol were added. The samples were frozen at -80°C for 2h then centrifuged at 3200 x g at 4°C for 45min and washed with 10ml of 70% ethanol. After air drying the pellets, they were resuspended in 150ml of 10mM Tris-HCl, pH 8. Ligation efficiency was determined by running 5ml of 3C material on a 0.6% agarose gel, and a significant upward shift in the digested DNA should be observed. To generate the 4C library from the 3C material, the 3C library was digested with 50U of MboI (NEB R0147L) in a 500ml reaction overnight. A 10ml aliquot was removed from the reaction to determine digestion efficiency on a 1.5% agarose gel. If digestion was successful (the DNA appears as a smear of fragments smaller than 1000bp), the reaction was heat inactivated for 20min at 65°C. Next, samples were transferred to a 50ml tube to which 1.4ml of 10x T4 DNA ligase buffer, 200U of T4 DNA ligase, and water up to 14ml were added before ligation at 16°C overnight. The samples were then precipitated with 14ml of glycogen (Thermo Fisher Scientific R0561), 1.4ml of 3M sodium acetate (pH 5.4), and 35ml of 100% ethanol at -80°C for 2h. The samples were pelleted at 3200 x g for 45min and washed with 10ml of 70% ethanol before air drying and resuspending in 150ml of 10mM Tris-HCl, pH 7.5. The DNA samples were purified using the QIAquick PCR Purification Kit (Qiagen 28106) following the manufacturer's protocol, and the concentration was measured via nanodrop. To amplify the DNA template and generate the 4C library, 3 equivalent PCR reactions with 200ng of input 4C material were ran simultaneously using the Expand Long Template PCR System (Sigma-Aldrich 11681842001) and 5ml from each reaction was ran on a 1.5% agarose gel to verify the success of the reaction. All successful reactions were combined and purified using the High Pure PCR Product Purification Kit (Sigma-Aldrich 11732676001) and analyzed on an Agilent Tapestation using the D5000 ScreenTape, reagents, and ladder (Agilent 5067-5588, 5067-5589, 5067-5590, respectively). The 4C libraries were sequenced (75bp, single-end) on an Illumina NovaSeq 6000 instrument for a total of 1 million reads per sample.