GSM2344812: ∆RF3_minimal_ribosome; Escherichia coli; OTHER
Whole cell lysates were clarified by brief centrifugation. Ribosome footprints were created using Mnase digestion (45 enzyme units per absorbance unit of lysate at 260nm). Using sucrose gradients the monosome fraction of lysate was isolated and footprints were size selected and converted to a cDNA library. Total RNA was extracted from purified lysate for a simultaneous RNA-seq library production for total RNA samples. Small RNA and ribosomal RNA subtraction was performed in total RNA samples using MEGAClear (ThermoFisher) and MicrobExpress (ThermoFisher) kits. Subtracted total RNA was then subjected to alkaline fragmentation prior to library preparation. A Linker-1 adapter (IDT) was ligated onto the 3' end of size selected RNA fragments, either ribosome footprints or fragmented total RNA. A cDNA library was created using Superscript III (ThermoFisher). After gel purification of the cDNA library all ssDNA fragments were circularized using CircLigase (Epicentre). For ribosome profiling samples rRNA containing circles were depleted using oligo-biotin  and streptavidin coupled Dynabeads (ThermoFisher) subtraction. A PCR amplification off the circles using HF Phusion (NEB) completed the libraries by adding Illumina Tru-Seq adapters and a unique index for each sample.