Chromatin Immunoprecipitation and RNA-IP Protocols

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 h. ChIP was performed essentially as described12 (link). Briefly, cells were fixed with 1% paraformaldehyde 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 s ON/OFF cycles). Five microlitres 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. 56 (link)) were used for IPs. RIP experiments were performed essentially as described13 (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.