GSM5736215: Sample treated with 5nM PNA after INR-Seq – R1; Escherichia coli K-12; OTHER
To purify the RET-stalled monosomes, the associated transcripts were cleaved with MNase and the digested samples were subsequently run on sucrose gradients, as is common for bacterial Ribo-seq (Figure 1) (Mohammad et al., 2019). After collection of the 70S peak, RPFs were isolated using acidic phenol-chloroform extraction followed by size selection of 15 – 45 nt long RNA on a denaturing polyacrylamide gel. RNA 5' ends were phosphorylated with T4 polynucleotide kinase (New England Biolabs). Adapters were ligated to the 5' and 3' ends of the RNA and first-strand cDNA synthesis was performed using M-MLV reverse transcriptase. The cDNA was then amplified using a high fidelity DNA polymerase and purified with Agencourt AMPure XP beads (Beckman Coulter). The cDNA samples were pooled in equimolar amounts and the rDNA within the pool was depleted targeting 24 unique sequences (Table S1) using a CRISPR-Cas9 protocol similar to DASH (Prezza et al., 2020). The final pool was subjected to sequencing on an Illumina NextSeq 500 system using 75 nt single-end read length.