GSM8889037: SK-N-BE(2)c, NCA, VP2; Homo sapiens; OTHER
The 4C templates were prepared as described previously van de Werken et al. 2012 (Methods Enymol, 513, 89-112). In brief, 10 x 106 SH-SY5Y or SK-N-BE(2)c cells were seeded per template. The day after seeding or 48 hours after ASO transfection cells were detached by trypsinization, and cross-linked with 2% formaldehyde for 10 min at room temperature. Cell lysates were digested with 400 U DpnII, and proximal DNA fragments were ligated with 50 U T4 DNA ligase. Ligated DNA circles were de-crosslinked overnight using proteinase K and purified with NucleoMag P-Beads to obtain an intermediate 3C template. 3C templates were digested with 50 U Csp6I and religated as described before, resulting in 4C templates. Adaptor-containing reading and non-reading primers, specific to the viewpoints of interest, were designed to amplify interacting genomic regions relative to the viewpoint. For each viewpoint, 16 PCR reactions, with 200 ng 4C template as input, were pooled. Resulting 4C-sequencing libraries were purified using QIAquick PCR Purification kit. 4C-sequencing libraries were pooled, and 1.6 pM with 1% PhiX was loaded on an NextSeq 500 instrument (NextSeq 500/550 High-Output kit, 75 cycles) for single-end sequencing.