GSM5624617: cHiC__ESC_E14Tg2a_depletionCTCF_mouse_Rep-1; Mus musculus; Hi-C
GSM5624617
3C libraries were prepared from fixed nuclei as described previously (Kragesteen et al, 2018). Briefly, lysis buffer was removed by centrifugation at 400 g for 5 min at 4 °C, followed by supernatant aspiration, snap-freezing, and pellet storage at − 80 °C. Later, nuclei pellets were thawed on ice, resuspended in 520 μl 1× DpnII buffer, and then incubated with 7.4 μl 20% SDS shaking at 900 rpm. at 37 °C for 1 h. Next, 75 μl 20% Triton X-100 was added and the pellet was left shaking at 900 rpm at 37°C for 1 h. A 15-μl aliquot was taken as a control for undigested chromatin (stored at − 20°C). The chromatin was digested using 40 μl 10 U/μl DpnII buffer shaking at 900 rpm at 37°C for 6 h; 40 μl of DpnII was added and samples were incubated overnight, shaking at 900 rpm. at 37°C. On day three, 20 μl DpnII buffer was added to the samples followed by shaking for an additional 5 h at 900 rpm. at 37 °C. DpnII subsequently was inactivated at 65 °C for 25 min and a 50-μl aliquot was taken to test digestion efficiency (stored at − 20 °C). Next, digested chromatin was diluted in 5.1 ml H2O, 700 μl 10× ligation buffer, 5 μl 30 U/μl T4 DNA ligase and incubated at 16°C for 4 h while rotating. Ligated samples were incubated for a further 30 min at room temperature. Chimeric chromatin products and test aliquots were de-cross-linked overnight by adding 30 μl and 5 μl proteinase K, respectively, and incubated at 65 °C overnight. On the fourth day, 30 μl or 5 μl of 10 mg ml−1 RNase was added to the samples and aliquots, respectively, and incubated for 45 min at 37°C. Next, chromatin was precipitated by adding 1 volume phenol-chloroform to the samples and aliquots, vigorously shaking them, followed by centrifugation at 4,000 rpm at room temperature for 15 min. To precipitate aliquot chromatin, 1 volume 100% ethanol and 0.1 volume 3M NaAc, pH 5.6 was added and the aliquots placed at -80°C for 30 min. DNA was then precipitated by centrifugation at 5,000 rpm. for 45 min at 4°C followed by washing with 70% ethanol, and resuspension in 20 μl with 10 mM Tris-HCl, pH 7.5. To precipitate samples, extracted sample aqueous phases were mixed with 7 ml H2O, 1 ml 3M NaAc, pH 5.6, and 35 ml 100% ethanol. Following incubation at −20°C for at least 3 h, precipitated chromatin was isolated by centrifugation at 5,000 rpm for 45 min at 4 °C. The chromatin pellet was washed with 70% ethanol and further centrifuged at 5,000 rpm for 15 min at 4 °C. Finally, 3C library chromatin pellets were dried at room temperature and resuspended in 10 mM Tris-HCl, pH 7.5. To check the 3C library, 600 ng were loaded on a 1% gel together with the undigested and digested aliquots. The 3C library was then sheared using a Covaris sonicator (duty cycle: 10%; intensity: 5; cycles per burst: 200; time: 6 cycles of 60 s each; set mode: frequency sweeping; temperature: 4–7 °C). Adaptors were added to the sheared DNA and amplified according to the manufacturer's instructions for Illumina sequencing (Agilent). The library was hybridised to the custom designed SureSelect beads and indexed for sequencing (75–100 bp paired-end) following the manufacturer's instructions (Agilent).