GSM4905168: MED14-IAA7, MED17-FLAG, +IAA, NAPP1 Rep 1; Saccharomyces cerevisiae; ChIP-Seq
ChIP was performed as described (PMID 19378172) with minor modifications. A 100 mL culture was grown to an OD600 ≥ 0.8 and treated with 500 μM 3-IAA or an equal volume of DMSO for 15 min. After 15 min of 3-IAA or DMSO treatment, the culture was split into two flasks. One culture was treated with 6 μM 1-Napthyl PP1 (NAPP1, Cayman Chemicals) and the other was treated with an equal volume of DMSO for 15 min. Cells were crosslinked using 1% formaldehyde for 30 minutes at 30C and then quenched using Tris pH 8.0 (final concentration of 0.75 M). Crosslinked cells were lysed with bead beating using a Disruptor Genie for 8 cycles of 3 min at 2850 rpm and 1 min on ice. Then, lysate was transferred to a new tube and centrifuged as previously described. Lysate was sonicated using a Bioruptor Pico for 8 cycles of 30 sec on and 30 sec off. Soluble chromatin was immunoprecipitated using 30 μL of serum bovine albumin pre-blocked Sigma FLAG M2 magnetic beads (catalog number M8823) overnight at  4C. Beads were washed as described and and decrosslinked by resuspending the beads in 98 μL of TE/1% SDS and shaking (800 RPM) at 65C for 12 -16 hours. DNA was purified from the supernatant using a Qiagen PCR purification kit per the manufacturer's protocol. Sequencing libraries were constructed by the Indiana University Center for Genomics and Bioinformatics (CGB) using the NEBNext Ultra II Library Prep Kit for Illumina and sequenced for 80 cycles in paired-end mode on the Illumina NextSeq 500 platform at the CGB. NEB Next Ultra II DNA Library Prep Kit for Illumina