title: GSM2256653: Sample 5_RNAseq experiment rep1; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: Extracts were prepared as previously described (Ingolia et al., 2009, Science, PMID: 19213877), except that the frozen cells were pulverized with a mixer mill at 30 Hz. The total amount of RNA in the extracts was quantified using RiboGreen (Invitrogen), and then, 50 ng of total RNA was diluted to 300 µL with the lysis buffer. The sample was subjected to preparation of ribosome footprints according to a previously described method (Ingolia et al., 2012, Nat Protoc, PMID: 22836135). Briefly, total RNA was treated with RNase I (Epicentre), and then the ribosomal pellet was collected by sucrose cushion centrifugation. RNA was recovered from the pellet with TRIzol (Life Technologies) and purified with Direct-zol RNA MiniPrep (Zymo), followed by isopropanol precipitation. RNA-seq libraries were generated using TruSeq Standard Total RNA Library Prep Kit (Illumina) from total RNA prepared as described above.