GSM8764088: Myc_ESC_DMSO_4C-seq_rep1; Mus musculus; OTHER
The cell pellets were washed twice in PBS and resuspended in 300 μL of lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630, and 1X cOmplete EDTA-free protease inhibitor cocktail) and incubated on ice for 20 min. Following centrifugation at 500 × g for 5 min at 4˚C, the pellet was resuspended in 50 μL of 0.5% SDS and incubated at 65˚C for 10 min on a shaker at 1,250 rpm. SDS was quenched by adding 150 μL of ddH2O and 25 μL of 10% v/v Triton X-100 at 37˚C for 15 min on a shaker at 1,250 rpm. To initiate the first digestion, 25 μL of NEBuffer DpnII and 10 µL of DpnII enzyme were added, and the samples were incubated overnight at 37˚C on a shaker at 750 rpm. The following morning, an additional 2 µL of DpnII was added, and the samples were incubated for 2 hr. Digestion efficiency was confirmed using gel electrophoresis. The enzyme was then inactivated by incubating at 65˚C for 20 min. Once the samples cooled down, 645 µL of ddH2O, 120 µL of 10X T4 DNA ligase reaction buffer, 120 µL of 10% v/v Triton X-100, 12 µL of 10 mg/mL BSA, 1 µL of 2000 U/µL T4 DNA ligase, and 60 µL of 10 mM ATP were added to the samples. The ligation reaction was incubated overnight at 16˚C in a water bath in a cold room. The following morning, 0.5 µL of 2000 U/µL T4 DNA ligase and 30 µL of 10 mM ATP were added, and the samples were incubated at RT with rotation until the ligation efficiency was verified using gel electrophoresis. After confirming successful ligation, 60 µL of 20 mg/mL Proteinase K and 120 µL of 10% v/v SDS were added, and the samples were incubated at 65˚C for 3 min. Then, 130 µL of 5 M NaCl was added, and the samples were incubated overnight at 65˚C on a shaker at 5,000 rpm for reverse crosslinking. The following morning, 10 µL of 10 mg/mL RNase A was added, and the samples were incubated at 37˚C for 1 h. DNA was purified using phenol-chloroform extraction and ethanol precipitation, and the pellet was dissolved in 100 µL of 10 mM Tris-HCl pH 8.0. The second digestion was performed by adding 20 µL of 10X Buffer B, 10 µL of 10 U/µL Csp6I, and 80 µL of ddH2O, and incubating overnight at 37˚C on a shaker at 700 rpm. After confirming digestion efficiency the next morning, a second ligation was performed by adding 2,204 µL of ddH2O, 300 µL of 10X T4 DNA ligase buffer, 1 µL of 2000 U/mL T4 DNA ligase, and 300 µL of 10 mM ATP. The reaction was incubated overnight at 16˚C in a water bath in a cold room. DNA was purified using phenol-chloroform extraction and ethanol precipitation, resulting in the 4C template. Primers for 4C libraries were designed around the transcription start site of each gene of interest according to previously described criteria.60 Libraries were generated from the 4C template using the inverse PCR strategy. Briefly, the first round of PCR was performed in quadruplicate per sample, with 150 ng of 4C template used for each reaction in the quadruplicate, employing Expand Long Template PCR system. The PCR products were pooled to increase the library complexity. A second round of PCR was performed using the initial PCR library as a template, with overlapping primers to add P5/P7 sequencing primers and sample-specific indexes. SPRIselect beads were used for DNA size selection throughout the protocol.