NextSeq 500 paired end sequencing; ChIP-seq analysis of yeast Ty1 integrase reveal the role of the AC40 interacting domain in the recruitment at both Pol I and Pol III-transcribed genes
ChIP-seq analysis of yeast Ty1 integrase reveal the role of the AC40 interacting domain in the recruitment at both Pol I and Pol III-transcribed genes
Yeast strain LV1689 was transformed with pCM185 expressing 3xHA-IN1-EPEA WT or harboring IN1 K617A, S621A or L622A mutations. ChIP was performed as previously described (Harismendy et al, 2003), with minor modifications. Briefly, log-phase cultures (50 ml) in SC-TRP to maintain plasmid selection were cross-linked with 1% formaldehyde for 5 min at room temperature.  Cells were pelleted by centrifugation and lysed with glass beads using an orbital shaker (IKA; VRX basic Vibrax; 2200 rpm, 40 min, 4°C). The cell lysate was drawn off the beads and spun for 20 min at 16000 rpm in a microcentrifuge at 4°C. The chromatin lysate was resuspended for 1 hour at 4°C on a rotative wheel and sonicated for 5 cycles (40 sec ON at high level and 20 sec OFF) in a Bioruptor (Diagenode; Denville, NJ, USA). After 1 hour at 4°C on a rotative wheel, the solubilized chromatin was recovered by centrifugation for 15 min at 9 000 rpm at 4°C in a microcentrifuge. The preparation of magnetic beads, immunoprecipitation with anti-HA 12CA5 antibodies, elution from beads and reversal of crosslinking were performed as described (Harismendy et al, 2003). Immunoprecipitated DNA was purified using a QIAquick PCR Purification Kit (Qiagen). Immunoprecipitated DNA has been sheared and ligated with illumina adapters.
Experimental Factor: genotype
CEN AmpR TRP1 pTetO7 3HA-IN5
Experimental Factor: immunoprecipitate
anti-HA