Fig. 1

Carbon copy-chromatin immunoprecipitation (2C-ChIP) combines ligation-mediated amplification and next-generation sequencing to measure co-immunoprecipitated DNA from given genomic regions. a Overview of the 2C-ChIP method. Chromatin is first immunoprecipitated with desired antibodies, and the resulting purified genomic DNA or corresponding input is used to produce a 2C-ChIP library by annealing and ligating 2C-ChIP primers in a multiplex setting. Resulting libraries are then PCR-amplified with primers featuring barcodes and processed for sequencing. b Bottom right insert highlights primer design in 2C-ChIP analysis. Genomic regions are detected by ligating Forward and Reverse 2C-ChIP primers annealed to the same DNA strand. Note that Reverse primers must be 5’end-phosphorylated for ligation to occur. Primer design shown here is for sequencing on Ion Personal Genome Machine™ (PGM™) system (Thermo Fisher Scientific) but can be adapted for sequencing on any platform. Forward 2C-ChIP primers include a universal sequence of choice (here a T3 complementary sequence; T3c) at their 5′ ends. Reverse 2C-ChIP primers are 5′-phosphorylated and feature the complementary PGM™ P1-key sequence (P1-key-c) destined to bind the Ion Sphere Particles (ISPs) used in sequencing. The libraries are then PCR-amplified with “2C-ChIP library amplification primers” to incorporate barcodes. The Forward amplification primer includes – 5′ to 3′ – an A-key sequence used in the PGM™ system, the barcode (BC), and a T3 sequence. The Reverse amplification primer is the P1-key sequence. Sequencing will occur from the A-key sequence such that barcodes will be read first