ChIP-exo Analysis of Pol II Binding

ES cells were grown and transfected with shRNA vectors as described above. Two independent transfection experiments were performed for each shRNA vector. Following a 10 min fixation with 1% formaldehyde in ES cell culture medium, chromatin was prepared from 5 to 10 million cells and sonicated as described^{32 (link)}. ChIP-exo experiments were carried out essentially as described^{33 (link)}. This included an immunoprecipitation step using antibodies against Pol II (sc-899, Santa Cruz Biotechnology) attached to magnetic beads, followed by DNA polishing, A-tailing, Illumina adaptor ligation (ExA2), and lambda and recJ exonuclease digestion on the beads. After elution, a primer was annealed to EXA2 and extended with phi29 DNA polymerase, then A-tailed. A second Illumina adaptor was then ligated, and the products PCR-amplified and gel-purified. Sequencing was performed using NextSeq500. Uniquely aligned sequence tags were mapped to the mouse genome (mm9) using BWA-MEM (version 0.7.9a-r786)^{34 (link)}. The uniquely aligned sequence tags were used for the downstream analysis.