title: GSM3692358: Scn5a_P1_2_del3; Mus musculus; OTHER
library_construction_protocol: 4C templates were prepared as previously described (van de Werken et al., 2012). 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 (van de Werken et al., 2012) using reading primers (Scn5a_P1+2 – GTCCCAAGGGCACACTGATC and Scn5a_enh1 – GAGACCCACACGTTAGGATC) as 4C viewpoints combined with non-reading primers (Scn5a_P1+2 – CCTCTAGAGAGCCTAGTCCC and Scn5a_enh1 – ACGGTGAGGACAACATAGAC). The primers were extended with Illumina adapter sequences and the samples were sequenced on the Nextseq.
attributes.tag: GEO Accession
attributes.value: GSM3692358