Ribo-Seq of E. coli strain BW25113: WT, replicate 2
E. coli strains were grown in LB at 37C to log phase (OD600nm=0.4). Deviating from this, JW4107 /+pBAD33_efpL_His6 was cultivated in LB supplemented with 30 g/ml chloramphenicol and 0.2 % (w/v) L(+)-arabinose.  Subsequently all samples were harvested by rapid filtration. Cells were scraped from the filter in aliquots for Ribo-seq and for RNA-seq and frozen in liquid nitrogen.  Total RNA was extracted from RNA-seq aliquots in trizol before mRNA was rRNA depleted, fractionated, and converted into Illumina compatible cDNA libraries.  Ribo-seq aliquots were lysed in ice-cold polysome lysis buffer by bead beating in a FastPrep-24 with Cool Prep Adapter. Lysates were clarified by centrifugation and ribosomes were subsequently pelleted by ultracentrifugation and resuspended in polysome digestion buffer. Samples were then digested with MNase. Ribosome protected mRNA fragments were purified and selected to have the length between 20-40 nt on 15 % urea PAGE gels, contaminating rRNA was depleted from samples using custom biotinylated rRNA depletion oligos for E. coli. The enriched fragments were converted into Illumina compatible cDNA libraries.  Both stranded mRNA-seq and Ribo-seq libraries were sequenced 150PE on Illuminas Nova-seq 6000 platform to depths of 10 million and 30 million raw read pairs per sample respectively. Reads preprocessing, including removing the adapters and other technical sequences, duplicate sequences and reads shorter than 30nts, was performed using Cutadapt and custom python script. Reads from Ribo-seq and RNA-seq libraries that were mapped to rRNA and tRNA sequences (Bowtie v1.2) were subtracted. The reads were aligned to E. coli strain BW25113 genome (RefSeq accession number NZ_CP009273.1) with Bowtie using parameters (-m 1 -l 25 -n 2 -S).