4C of Sox2 locus in mouse embryonic stem cells
Single cell suspensions of mouse embryonic stem cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Nuclei were isolated and cross-linked chromatin was digested with DpnII. This was followed by proximity ligation, after which cross-links were reversed. A secondary restriction enzyme digestion was performed using NlaIII. Primers were designed such that the first read of a paired-end read would sequence the primer sequence derived from the bait region and read into the target region of interest. The primers were designed to include the Illumina adaptor sequences necessary for sequencing as well as the presence of barcodes derived from Illuminas TruSeq adaptors that allowed for multiplexing of 4C-seq reactions. Typically, 200 ng of the resulting 4C template was used for the subsequent PCR reaction. The 4C template was PCR amplified for 30 cycles and 3-4 reactions were pooled together. The PCR products were then purified using dual SPRI bead selection to get template between 120  1000bp (Beckman Coulter) according to the manufacturers instructions. The concentrations of each 4C library were calculated using the KAPA qPCR system and comparison to a standard curve. The libraries were then combined and sequenced on a Illumina HiSeq 4000 instrument with single-ended 50bp reads.