GSM5518520: E. coli Ribo-seq - ΔleuB Leu-lim 0.1 rep3; Escherichia coli; OTHER
The method of RNA extraction and ribosome profiling was described in detail previously (Li et al., 2018). After quantification of RNA concentration with NanoDrop, samples with 500µg RNA were digested with 750U MNase (10107921001, Roche) for 1 hour at 25˚C before being quenched with 6mM EGTA. The lysates were then layered onto a 10%-55% sucrose gradient and centrifuged. The monosome fraction was collected and snap frozen in liquid nitrogen. There were no observed polysome peaks, which indicated thoroughgoing digestion. The RNA was separated using hot phenol and size selected on 15% TBE-Urea PAGE gels run for 1 hour at 210V. Gels were stained with SYBR Gold and visualized using Dark Reader (Clare Chemical Research). Finally, for ribosome profiling, RNA fragments with size between 25-40 nt were extracted using isopropanol precipitation. RNA fragments from footprints were dephosphorylated at the 3' end by PNK (M0201, NEB). The repaired fragments were linked to the Universal miRNA Cloning Linker (S1315S, NEB), reverse transcribed (18080044, Thermo), and circularized (CL4111K, Epicentre). rRNA was subtracted from the circularized samples before PCR amplification (M0531L, NEB) and size selection. High quality PCR samples were checked by Bioanalyzer highly sensitive DNA chip.