title: GSM3439131: 4C_MEF2C_recVP7_SHSY5Y_rep1; Homo sapiens; OTHER
library_construction_protocol: 4C templates were prepared according to the protocol by Van de Werken et al. (Methods Enzymol., 2012). In brief, for each template 1x10^7 cells were detached, counted, resuspended and crosslinked by incubating them with 2% formaldehyde for 10 min at room temperature. Following cell lysis, crosslinked DNA was digested with 400U of DpnII restriction enzyme (NEB #R0543L) and nearby DNA fragments were ligated using 50U of T4 DNA ligase (Roche #10799009001). Ligated DNA circles were de-crosslinked overnight using proteinase K and purified with NucleoMag P-Beads (Macherey-Nagel) to obtain an intermediate 3C template. A second round of digestion and ligation, using 50U of Csp6I restriction enzyme (Thermo Scientific #ER0211), resulted in 4C templates. Adaptor-containing reading and non-reading primers, specific to the viewpoints of interest, were designed to amplify all captured, interacting DNA fragments. For each viewpoint, 16 PCR reactions, each using 200 ng of input 4C template, were pooled. Resulting 4C sequencing libraries were purified using High Pure PCR Product Purification kit (Roche #11732676001) and QIAquick PCR Purification kit (Qiagen #28106). Approximately 15-20 different 4C sequencing libraries were pooled and sequenced simultaneously on an Illumina NextSeq 500 (single-end, 75 nt, loading concentration 1.6 pM).
attributes.tag: GEO Accession
attributes.value: GSM3439131