GSM1637976: Puf6p_targets; Saccharomyces cerevisiae; OTHER
Cells were lysed in 1XPBS, 0.5% NP-40, 1X Complete Mini Protease Inhibitor, EDTA-free and cleared by sequential spins at 1,300Xg for 5 min and 20,000Xg for 10 min at 4°C. Clarified lysate was passed through a Costar Spin-X filter (Corning CLS8160), mixed with RNase T1 (Fermentas EN0541) to 1 U/µL, and incubated at 22°C for 15 min followed by 5 min incubation on ice. Lysate was then directly mixed with IgG magnetic beads (prepared by coupling rabbit IgG (Sigma-Aldrich I5006) to Dynabeads M-270 Expoxy (Invitrogen 143-01)) to pull down Puf::TAP. RNase T1 digestion, CIP treatment, and 3' DNA linker ligation were performed as described for Puf3p in Freeberg et al. (Gen Biol 2013). Afterwards, 5' end phosphorylation was performed on-bead in 20 µL of PNK mix (70 mM Tris pH 7.6, 10 mM MgCl2, 5 mM DT, 1 µL P32 rATP (6000 Ci/mmol 10 mCi/mL Perkin Elmer BLU502Z500UC), 1 U/µL T4 polynucleotide kinase) and incubated at 37°C for 15 min. 2 µL of 10 mM ATP was added to the mix, and the reaction was incubated for 10 min. After SDS-PAGE and transfer, crosslinked RNAs were visualized by autoradiography, and the corresponding Puf band was excised. Library construction was carried out as described for Puf3p in Freeberg et al. (Gen Biol 2013), omitting the 5' end phosphorylation step.
GEO Accession
GSM1637976