GSM5737215: CTCF (Δ5|6:6|7) MN rep3; Mus musculus; OTHER
Cells were processed for 4C-seq as described in Narendra et al., 2015; following the protocol details as published in van de Werken et al., 2012. Cells were trypsinized, counted, and 1 x 107 cells were crosslinked with the Crosslinking Solution (2% formaldehyde and 10% FBS in 1X PBS) for 10 min at room temperature. After the reaction was quenched with glycine, the cells were lysed on ice with 1ml Lysis Buffer (50mM Tris pH 7.3, 150mM NaCl, 5mM EDTA, 0.5% NP-40, and 1% Triton X-100) for 15 min. Nuclei were spun down and frozen at -80 oC. Upon thawing on ice, nuclei were resuspended in 360ul H2O. The primary restriction enzyme was DpnII. The secondary restriction enzyme was Csp6I. 1ug 4C template DNA obtained was amplified using inverse primers containing barcoded illumina sequencing adapters on either end. Libraries were prepared according to manufacturer's instructions (Illumina) as described in Narendra et al., 2015. 4C-seq libraries were constructed by attaching barcoded Illumina adapters to the 5' end of the primer. PCR reactions were performed using the Expand Long Template PCR System (Roche) and about 100-700bp DNA was gel purified and quantified prior to sequencing.