P-GSE21992-2 - P-GSE21992-2
nucleic acid library construction protocol
mRNA-Seq: poly(A)-selected RNA was randomly fragmented by partial alkaline hydrolysis. Size-selected RNA fragments (25-45 nt) were used for library preparation. Ribosome profiling: Cell extracts were digested with RNase I for 30 min at room temperature, and monosomes were purified by sucrose gradient centrifugation. Size-selected RNA fragments (~27-33 nt) were used for library preparation. Library preparation: Libraries were prepared as in Grimson et al, 2008 (GSE12578) but with the following modifications. Because RNase I-digestion and alkaline-fragmentation products terminate with a 5'-hydroxyl and a 3'-phosphate, they were 3'-dephosphorylated before ligation to the 3' adaptor. Gel-purified 3'-ligation products were then 5'-phosphorylated before the 5'-ligation step.