GSM5006210: rep2 ribo-seq araM; Escherichia coli; OTHER
Cells were collected via vacuum filtration, flash frozen in liquid nitrogen, ground to a fine powder with frozen lysis buffer, then allowed to thaw to complete lysis. Lysis buffer contained 20 mM Tris pH 8, 10 mM MgCl2, 100 mM NH4Cl, 5 mM CaCl2, 1 mM chloramphenicol, 0.1% v/v sodium deoxycholate, 0.4% v/v Triton X-100, 100 U/mL DNase I, 1 uL/mL SUPERase-In (Thermo Fisher Scientific AM2694) Each sample produced a matched set of RNA-seq and ribo-seq fragments which were size selected to a range of 18-50nt. These were ligated with a 5' linker (containing a 4nt UMI) and a 3' linker (containing a 5nt UMI and a 5nt sample barcode). rRNA depletion was completed with the Illumina Ribo-Zero rRNA Depletion Kit for bacteria. RNA-seq and ribo-seq in E. coli cell lines from the Long Term Evolution Experiment