4C-seq to detect the interaction of the EVI1 promoter (viewpoint of the experiment) with chromatin regulatory elements in a selection of ovarian cancer cell lines and other cancer cell lines
4C-seq to detect the interaction of the EVI1 promoter (viewpoint of the experiment) with chromatin regulatory elements in a selection of ovarian cancer cell lines and other cancer cell lines
10 million cells were harvested for one 4C-seq experiment. First, cells were washed once with 10 mL DPBS and resuspended in 5 mL DPBS/10 % FBS.  All cells were maintained in a humidified atmosphere at 37°C with 5% CO2. COV-434 cells were cultured in DMEM, 2 mM Glutamine and 10% FBS. ES-2 cells were cultured in McCoy's 5a and 10% FBS. Kuramochi and SKOV-3 cells were cultured in RPMI 1640 and 10% FBS. OAW28 and OAW42 cells were cultured in DMEM, 2 mM glutamine, 1 mM NaP, 5 mg bovine insulin and 10% FBS. OVISE cells were cultured in DMEM and 10% FBS. TOV-112D cells were cultured in 1:1 MCDB 105 medium plus Medium 199 and 15% FBS. Fixation and lysis of cells 10 million cells were harvested for one 4C-seq experiment. First, cells were washed once with 10 mL DPBS and resuspended in 5 mL DPBS/10 % FBS. Cells were fixed with 5 mL of 4 % formaldehyde in DPBS/10 % FBS for 10 min at RT on an orbital mixer. The fixation was quenched by adding 1.425 mL 1 M glycine solution. The supernatant was removed after centrifugation for 8 min at 368° rcf and 4 °C. The cell pellet was resuspended in the remaining supernatant and transferred to a 1.5 mL tube. The supernatant was completely removed after another centrifugation for 8 min at 368°rcf and 4 °C. Cells were then resuspended in 1 mL lysis buffer and incubated at RT for 5 min, followed by incubation at 65 °C for 5 min. Lysed cells were harvested by discarding the supernatant after centrifugation at 300 rcf for 5 min at 4 °C, followed by washing once with 1 mL PBS. After centrifugation at 600 rcf for 2 min at 4 °C, the supernatant was removed and the pellet was snap-frozen in liquid nitrogen and stored at -80 °C.  First digestion and first ligation The restriction enzyme DpnII was used as the first cutter. The frozen pellet was resuspended in a solution consisting of 440 µL water, 60 µL restriction enzyme buffer and 15 µL of a 10 % SDS solution. This mixture was incubated at 37 °C for 1 h with shaking at 900 rpm. In between, the reaction was mixed by pipetting approximately every 10 min. Then, 75 µL of 20 % Triton X-100 was added and the mixture was incubated for 1 h at 37 °C with shaking at 900 rpm. To start digestion, 4 µL (200 U) DpnII (50,000 U/mL) was added and incubated for 4 h at 37 °C with shaking at 900 rpm. Subsequently, another 4 µL of the enzyme was added and incubated at 37 °C o/n with shaking at 900 rpm. The next morning, 4 µL DpnII was freshly added and samples were incubated for an additional 4 h at 37 °C with shaking at 900 rpm. A total of 600 U of DpnII per sample was used for the first restriction digestion. The DpnII enzyme was heat-inactivated at 65 °C for 20 min, and the sample was transferred to a 50 mL tube. Ligation was started by adding 7 mL 1× ligation buffer and 10 µL (50 U) T4 Ligase (5 U/µL). The sample was incubated o/n at 16 °C.  Reverse cross-linking and DNA precipitation 15 µL Proteinase K (20 mg/mL) was added to the ligated sample and incubated o/n at 65 °C. The next day, 30 µL RNase A (10 mg/mL) was added and the sample was incubated at 37 °C for 45 min. For DNA precipitation, 7 mL phenol-chloroform was added, mixed vigorously, and centrifuged at 3,066 rcf for 15 min at RT. The resulting DNA-containing aqueous phase, was transferred to a new 50 mL tube to which 7 mL water, 7 µL glycogen, 1.5 mL 2 M NaAc pH 5.6, and 35 mL 100 % ethanol were added. This solution was mixed vigorously and stored at -80 °C until the sample was completely frozen (o/n or over the weekend). Thereafter, the sample was thawed on ice and centrifuged at 4,415 rcf and 4 °C for 45 min. The supernatant was removed, 10 mL of cold 70 % ethanol was added, and the sample was centrifuged at 3,066 rcf and 4 °C for 15 min. After removing the supernatant, the pellet was dried at RT and finally resuspended in 150 µL 10 mM Tris pH 7.5 at 37 °C.  Second digest, second ligation, DNA precipitation and purification 150 µL sample was digested with 5 µL (50 U) Csp6I (10 U/µL), 50 µL 10× Buffer B, and 295 µL water o/n at 37 °C.The restriction enzyme Csp6I was heat-inactivated at 65 °C for 25 min. The sample was transferred to 50 mL tubes along with 1.4 mL 10× ligation buffer, 20 µL (100 U) T4 Ligase (5 U/µL), and 12 mL water for a second ligation o/n at 16 °C. The following day, 1.4 mL 2 M NaAc pH 5.6, 14 µL glycogen, and 35 mL 100 % ethanol was added and the sample was frozen at -80 °C at least o/n. Next, the sample was thawed on ice and centrifuged at 3,066 rcf and 4 °C for 45 min. The supernatant was removed and 15 mL ice-cold 70 % ethanol was added. The sample was centrifuged again at 3,066 rcf and 4 °C for 45 min, and the supernatant was discarded. The precipitated DNA was air-dried and then dissolved in 150 µL 10 mM Tris pH 7.5 at 37 °C. DNA was purified with the QIAquick PCR Purification Kit using three columns (max. binding capacity 10 µg/column) per sample. Each column was eluted with 50 µL 10 mM Tris pH 7.5. The eluates from the three columns of a sample were pooled and the concentration was measured. The sample was stored at -20 °C until further processing. PCR was performed using the Expand Long Template PCR System with the followign protocol: 2 min at 94 °C, 35 cycles at: 94 °C for 15 s, 55 °C for 1 min and 68 °C for 3 min, and one final cycle at 68 °C for 7 min. One of eight forward primers (IDs 78−85, Supplemental table xy) with variable 2-nt barcodes and one universal reverse primer (ID 86) were used. Barcoding of samples allowed pooling of multiple 4C samples on a single sequencing lane. In general, 100 ng of 4C DNA was used for all cell lines screened and pooled in a maximum of 8 samples. The resulting libraries were purified using the High Pure PCR Product Purification Kit and an elution volume of 50 µL 10 mM Tris pH 8.0. Libraries were subsequently pooled and the concentration was measured. Cell lines were sequenced in four different multiplexes. For information of the submissions and the cell lines and primers used, please see Supplementary Table xy.