GSM3553914: WT_ribo_rep2; Escherichia coli; OTHER
When the culture density reached A600 of ∼0.5 cells were harvested by rapid filtration, frozen in liquid nitrogen, and processed for ribosome profiling as described in [Becker, A.H., Oh, E., Weissman, J.S., Kramer, G. & Bukau, B. Selective ribosome profiling as a tool for studying the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes. Nat Protoc 8, 2212-39 (2013)]. For the preparation of RNA-seq samples, total RNA was isolated from the cell lysates, short RNA and rRNA were subtracted using Ambion MEGAclear kit and MICROBexpress bacterial mRNA enrichment kit, respectively, and RNA was fragmented using the RNA Fragmentation Reagents kit from Ambion. The library for next-generation sequencing was prepared using RNA fragments ranging in size from 20 to 45 nt. During data analysis, the first position of the P site codon was assigned at 15 nt away from the 3' end of the read.