3 protocols
nucleic acid extraction protocol
Cell lysates were partially clarified at 4,000 rpm and digested with RNAse A (Sigma). TAP-tagged proteins were pulled-down with Rabbit IgG conjugated Dynabeads (Invitrogen) and washed in lysis buffer with 1M urea to reduce non-specific binding. The RNA for the control is extracted using glass bead lysis and phenol/chloroform extraction. mRNA is then selected by oligo(dT) pull-down. For the rest of the samples (Dhh1, Lsm1, Pat1 and Sbp1), RNA is covalently cross-linked to protein using UV-light and pulled-down with the protein via a TAP-tag purification protocol. The protein is then removed from the RNA using proteinase K. This procedure has been done according to standard CLIP-seq protocols and is described in further detail (buffers etc.) in Mitchell et al. 2013. RNA fragments were isolated, decapped and cloned into RNA libraries as in Ule et al. Methods 2005 and Wang et al. Methods 2009
growth protocol
Strains were grown in YEPD at 30° C to mid-log.
sample treatment protocol
Cells were re-suspended in PBS for 10 minutes to induce P-bodies. Stressed cells were UV-crosslinked at 0.8–1.2 J/cm2.