4C templates were prepared as previously described (de Krijger et al., 2020). In short, snap frozen adult mouse hearts of Scn5a RE6-9 mutant mice and wildtype littermates were crushed, dissociated and dounce homogenized to obtain single cell suspension. Chromatin was cross-linked with 2% formaldehyde in PBS with 10% FCS for 10 minutes at room temperature. Nuclei were isolated and cross-linked DNA was digested with DpnII, followed by proximity ligation. Cross-links were removed and a secondary restriction digestion with Csp6I performed, followed again by proximity ligation. Per viewpoint, 8 PCR reactions were performed on 200 ng of 4C template each, to a total of 1.6 µg. PCR reactions were pooled and purified for next generation sequencing. The 4C-seq PCRs were essentially performed as previously described (de Krijfer et al., 2020) using reading primers (CTCF left – AAAAGCTCAGAGTGGAGATC and Nppa promoter – GCCCTAGGAGAGCTATGATC) as 4C viewpoints combined with non-reading primers (CTCF left – ATTCTACTGATGCTGCATGG and Nppa promoter –TAACAGGGACATCTAGGGTG). The primers were extended with Illumina adapter sequences and the samples were sequenced on the Nextseq.
GEO Accession
GSM4696227