Purelink rna micro columns

Immunoprecipitation of TAP-tagged proteins was carried out using monoclonal antibodies against protein A (Sigma), and myc-tagged proteins were purified using the 9E11 monoclonal antibody (Abcam). For Scw1-TAP and Zfs1-TAP RIp-chips were performed as described [62] (link) except for the following modifications: 1) the lysis buffer contained 1 mM PMSF and 1∶100 protease inhibitor cocktail (sigma P8340) and 2) magnetic beads containing the immunoprecipitate were resuspended in 50 µl of wash buffer containing 1 mM DTT, 1 unit/ml of SuperaseIN (Ambion) and 30 units/ml of AcTev protease (Life Technologies). The solution with the beads was incubated for 1 h at 19°C, the supernatant recovered and RNA extracted using PureLink RNA micro columns (Life Technologies) according to the manufacturer's instructions. The RNA was eluted from the column in 12 µl and used for labelling without amplification. For Red1-myc we followed a published protocol [62] (link), except that the lysate was prepared in the following buffer: 10 mM Hepes pH 7.4, 100 mM KCl, 5 mM MgCl₂, 25 mM EDTA, 0.5% NP-40, 1% Triton X-100, 0.1% SDS and 10% glycerol containing 1 mM PMSF and 1∶100 protease inhibitor cocktail (sigma P8340).

For RPF analyses, preparation of cell extracts, RNase treatment, separation of samples by centrifugation through sucrose gradients, and isolation of protected RNA fragments were performed as previously described^{21 (link)}. Gel purified RNA fragments were treated with 10 units of T4 PNK (Thermo Fisher) in a low pH buffer (700 mM Tris pH 7, 50 mM DTT, 100 mM MgCl₂) for 30 min at 37 °C. ATP and buffer A (Thermo Fisher) were then added for an additional 30 min incubation. RNA fragments were column-purified (PureLink RNA micro-columns, Life Technologies). 100 ng were used as input for the NEXTflex Small RNA Sequencing Kit v2 (Bioo Scientific), and libraries were generated following the manufacturer’s protocol. For mRNA analyses, total RNA was isolated as previously described^{21 (link)}. rRNA was then removed from total RNA using Ribo-Zero Gold rRNA Removal Kit Yeast (Illumina), with 4 µg as input. Finally, 30 ng of rRNA-depleted RNA was used as starting material for the NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific).