ChIP and RNA-IP of Mmi1 in Fission Yeast

Cells were grown to mid-log phase at 32°C in YES. For phosphate starvation experiments, cells in mid-log phase were washed twice in dH₂O before being grown in PMG (−PO₄) for 4 hrs. ChIP was performed essentially as described^{12 (link)}. Briefly, cells were fixed with 1% paraformaldehyde (PFA) for 15 min at room temperature. Cells were lysed by bead beating (Biospec Prodcutes) and sonicated using a Bioruptor (Diagenode) sonicator at 5°C on high for a total of 20 min (30 sec ON/OFF cycles). 5 μL of Rpb1 antibody (#2629; Cell Signaling), 2 μL GFP antibody (G10362; Life Technologies), 2 μL H3 antibody (ab1791; Abcam), and 1 μL of H3K9me2 antibody (m5.1.1; ref. 55 (link)) were used for IPs. RNA immunoprecipitation experiments were performed essentially as described^{13 (link)}. Hisx6-TEV-Protein A-tagged Mmi1 was captured from cell lysate with IgG Dynabeads® (Life Technologies). Mmi1-bound RNA was isolated by phenol-chloroform extraction, DNase treated, and reverse transcribed. Quantitative analysis was performed by qPCR.