NextSeq 500 sequencing; 4C Sequencing using the Fos gene promoter as viewpoint in mouse Barrelette neurons at different time points during development
4C assays using wild-type brainstem tissue were performed as described previously (Splinter et al., 2012) with minor modifications. Two million of dissociated cells were cross-linked for 10 minutes with 2% paraformaldehyde, quenched with glycine and lysed in 50 ml lysis buffer (10mM Tris pH 7.5, 10mM NaCl, 2% NP-40, 1X protease inhibitors) for 30 minutes. Not applicable (no nucleic acid extraction is performed in 4C-seq). Nuclei were then digested with 800U NlaIII enzyme (NEB, R0125) followed by 8 hours 'in nuclei' ligation at 16˚ C with 2000U T4 DNA Ligase (NEB, M0202) (Nagano et al., 2015). Reverse crosslinked and purified DNA was further digested with 50U DpnII enzyme (NEB, R0543), followed by circularization. 3200ng of 4C library was amplified with bait-specific inverse primers (read Primer (NlaIII), amplification Primer (DpnII)), pooled and purified.