title: GSM2024749: R3_Na+; Saccharomyces cerevisiae; RIP-Seq
library_construction_protocol: Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with protease inhibitors) with FastPrep machine (MP Biomedicals). To dissolve large RNPs, the supernatants were treated with 50 U/ml RNase T1 (Fermentas) at 22°C for 15 min. Pull-downs were performed with streptavidin agarose beads (Thermo Fisher Scientific) in binding buffer (50 mM NaPi pH 8.0, 300 mM NaCl, 6 M GuHCl, 0.5% Tween-20). The second RNase T1 digestion was performed on the beads with a final concentration of 1 U/μl. Radiolabeling of RNA was performed by adding 0.5 µCi/µl γ 32P-ATP (Hartmann analytic) and 1 U/µl T4 PNK (New England Biolabs). To purify RNA, proteins were digested using 1.2 mg/ml proteinase K (Roche) in 2 x proteinase K buffer (100 mM Tris HCl pH 7.5, 200 mM NaCl, 2 mM EDTA, 1% SDS) for 30 min at 55˚C. The RNA was subsequently isolated using phenol-chloroform-isoamyl alcohol (125:24:1) (Sigma-Aldrich) as described (Schmitt et al., 1990). Purified RNA was subjected to 3’ and 5’ adapter ligation following Illumina’s TruSeq Small RNA Library Prep Guide. To reduce the rRNA species, RiboMinus transcriptome isolation kit (Invitrogen) was used according to the manufacturer's protocol. Reverse transcription using SuperScript III reverse transcriptase (Invitrogen), oligo dT and random hexamer was performed afterwards. The cDNA libraries were generated by a final PCR amplification step with llumina indexing primer.