Designing chip qpcr primers

Get the genomic coordinates either by

- Previous papers
- DNA sequence analysis (eg promoter analysis)
- Sequencing datasets (eg chip seq)

For this example, we are getting RNAP2 binding site at HIF1A promoter

1. download RNAP2 binding site data from ENCODE website (bed narrow peak file)
a. https://www.encodeproject.org/experiments/ENCSR564IGJ/
b. Filter view – bed narrow peak
c. Download default file hg38
2. Process in R
3. Export as excel
4. Get coordinates for your region of interest
a. Make sure this is at the start of canonical gene (look at ensembl, and crosscheck at
uniport)
5. Calculate 200 bp coordinate centred around the middle of the region of interest.
6. Download 200bp DNA sequence from UCSC website
7. Put into idt primer quest website and design ChIP qPCR primers (get 2 or 3 sets and test
them all on input and use the set with the best ct and dissociation curve)

Check RNApol2 coordinates

Check HIF1B coordinates

Check HRE sequences within primer region (snapgene)