GSM8497424: low_mg_rep1_rnaseq; Escherichia coli; RNA-Seq
Once the cultures reached an OD600 of ~0.4, they were split into two 50 ml conical tubes. Cells from one tube were pelleted and submitted to Genewiz for RNA-Seq, while the cells from the second tube were used for Ribo-RET. For Ribo-RET, cells were treated with retapamulin at a final concentration of 100 µg ml-1 for 5 minutes at 37°C with continuous shaking. After retapamulin treatment, cells were filtered through a nitrocellulose membrane with a pore size of 0.45 µm, quickly scraped off the surface, and flash-frozen in liquid nitrogen. The resulting samples were processed, and libraries were prepared as described previously (Cope et al. 2022) Libraries for RNA-Seq were prepared by Genewiz. Ribo-RET libraries were prepared as described previously (Cope et al. 2022) with the following modifications. Specifically, RNA fragments of 15-30 nucleotides (nt) in length were isolated by performing a tight gel size selection, and rRNA depletion was performed using the RiboCop for Gram-negative bacteria kit (Lexogen). The resulting RNA fragments were then pooled, and libraries were prepared, with a final library structure consisting of a 5' adapter – 4 random bases – insert – 5 random bases – sample barcode – 3' adapter. The randomized bases serve as Unique Molecular Identifiers (UMIs) for deduplication. The multiplexed library was sequenced on an Illumina HiSeq 4000 with a paired-end reading of 150 bases (PE150 runs) at a sequencing depth of 40-60 million raw reads per sample