GSM7018219: CLBGA_SOX11; Homo sapiens; OTHER
107 separated single cells that were cross-linked with 2% formaldehyde for 10 min at room temperature, cells were lysed. Cross-linked DNA was digested was digested using 200U of NlaIII (NEB #R0125S) as primary restriction enzyme. This was followed by proximity ligation using 50U of T4 DNA ligase (Roche #10799009001) and ligated DNA circles were de-cross-links overnight using proteinase K and purified with NucleoMag P-Beads (Macherey-Nagel, Düren, Germany) to obtain an intermediate 3C template. To generate the 4C template, DNA was redigested using 50U DpnII (NEB #0543S) as secondary antibody followed again by proximity ligation. Digestion and ligation efficiency were evaluated via agarose gel electrophoresis. Adaptor-containing reading and non-reading primers, specific to the viewpoints of interest, were designed to amplify all captured, interacting DNA fragments (SOX11 viewpoint reading = CCACCAAAATTTTCATCATG and non-reading = TCTTCTATGCATCCGATTCT; without adapter sequence). 4C-sequencing fragments were amplified using the Expand Long Template PCR System (Roche, 11681834001) and using High Pure PCR Product Purification kit (Roche #11732676001) and QIAquick PCR Purification kit (Qiagen #28106). Sequencing was performed on the NextSeq 500 platform (single-end, 75 nt, loading concentration 1.6 pM).