4C-seq assays were performed as previously reported27, 29–31. Whole embryos (9.5 days of development) or whole adult (8 weeks) mice brains (both strain CD-1; Charles River Labs) were processed to get approximately 10 million isolated cells, which were treated with lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.3% IGEPAL CA-630 (Sigma-Aldrich, I8896), 1X protease inhibitor cocktail (cOmplete, Roche, 11697498001)). Nuclei were digested with DpnII endonuclease (New England Biolabs, R0543M) and ligated with T4 DNA ligase (Promega, M1804). Subsequently, Csp6I endonuclease (Fermentas, Thermo Scientific, FD0214) was used in a second round of digestion, and the DNA was ligated again. Specific primers were designed near each gene promoter with primer3. Viewpoint fragend coordinates were as follows: mFTO: chr8:93837310–93837724; mIrx3: chr8:94325277–94326009. Illumina adaptors were included in the primers sequence32. 16 PCRs were performed with Expand Long Template PCR System (Roche, 11759060001) for each viewpoint, pooled together and purified using High Pure PCR Product Purification Kit (Roche, 11732668001). Quanti-iT™ PicoGreen dsDNA Assay Kit (Invitrogen, P11496) was used in order to measure sample concentration, and then they were sent for deep sequencing. 4C-seq data were analysed, with some changes, as described in 30.
