PCR enrichment

PCR enrichment is a recommended step in ChIP-exo to amplify the cDNA library. The amplification is carried out by a Pyrococcus-like enzyme called Phusion, which has a high-fidelity and robustness, and it requires the use of both qPCR and PCR thermocyclers. These are the settings for the qPCR thermocycler:

1. 98 °C for 2 minutes.

2. 99 cycles of:

   2.1. 98 °C for 15 seconds.

   2.2. 65 °C for 30 seconds.

   2.3. 72 °C for 30 seconds.

Once the amplification has been increased exponentially for 6-7 cycles, the sample should be moved to the PCR thermocycler, where the sample will be left for 10 minutes at 72 °C to perform the final elongation step. Afterwards, the sample should be moved to room temperature.