GSM5856232: SCR F1 mus rep1; Mus musculus musculus x Mus musculus castaneus; OTHER
Cells were fixed with 2% paraformaldehyde in 10% FBS in PBS for 10 min at 23°C. The fixation was quenched with cold glycine at a final concentration of 125 mM, then cells were washed with PBS and permeabilized on ice for 1 h with 10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.1% NP-40, and protease inhibitors. Nuclei were resuspended in DpnII restriction buffer at 10 million nuclei/mL concentration, and 5 million nuclei aliquots were further permeabilized by treatment for 1 h with 0.4% SDS at 37°C, then incubating for a further 1 h with 3.33% Triton X-100 at 37°C. Nuclei were digested overnight with 1500 U DpnII at 37°C, then washed twice by centrifuging and resuspending in T4 DNA ligase buffer. In situ ligation was performed in 400 μL T4 DNA ligase buffer with 20,000 U T4 DNA ligase overnight at 23°C. DNA was purified by reverse cross-linking with an overnight incubation at 65°C with proteinase K, followed by RNase A digestion, phenol/chloroform extraction, and isopropanol precipitation. The DNA was digested with 5 U/μg Csp6I at 37°C overnight, then re-purified by phenol/chloroform extraction and isopropanol precipitation. The DNA was then circularized by ligation with 200 U/μg T4 DNA ligase under dilute conditions (3 ng/μL DNA), and purified by phenol/chloroform extraction and isopropanol precipitation. For allele-specific 4C from the SCR-proximal bait, samples of the DNA were digested with BveI or Alw26I, cutting specifically at the region between the DpnII site and the 4C reading primer annealing site on the 129 or castaneus allele, respectively. For 129-specific 4C from the SCR bait, the material was digested with AvaIII, cutting specifically at the region between the Csp6I site and the 4C non-reading primer annealing site on the castaneus allele. There were no SNPs allowing castaneus-specific 4C from this bait. No digestion was required for allele-specific 4C from the human insertion sequence, since this was only present on the 129 allele. 100 ng aliquots of treated DNA were then used as template for PCR with bait-specific primers containing Illumina adapter termini. PCR reactions were pooled, primers removed by washing with 1.8× AMPure XP beads, then quantified on a Bioanalyzer (Agilent) before sequencing with a HiSeq 4000 (Illumina). 4C PCR products are quantified in a Bioanalyzer, and pooled in equimolar quantities for direct cluster generation on an Illumina HiSeq4000