GSM4296414: M2L_rep3; Pseudomonas fluorescens; OTHER
Per sample: 1.5 ml aliquots of mid-exponential phase culture were taken, and total RNA extracted (TRIzol Max Bacterial RNA isolation kit; Life Technologies). The YAMAT-seq method is an adaptation of that published for human cell lines (Shigematsu et al., 2017). For each sample, 5 ug total RNA was subjected to tRNA deacylation treatment by exposure to hydrochloric acid. Hybrid DNA/RNA Y-shaped adapters (Eurofins) were ligated to exposed 5'-CCA-3' tRNA ends using T4 RNA ligase 2 (New England Biolabs). Ligation products were reverse transcribed into cDNA (SuperScript III reverse transcriptase; Thermo Fisher), and amplified PCR (Phusion; Thermo Fisher). One of 27 sample-specific indices (Illumina) was added to each of the 27 reactions. The quality and quantity of each PCR product was checked using an Agilent DNA 7500 kit on a Bioanalyzer (Agilent), and samples combined in equimolar amounts into a single tube. The mixture was run on a 5 % polyacrylaminde gel (Bio-Rad Laboratories), and the group of bands ranging between 180 bp and 250 bp excised. DNA was extracted in deionized water overnight, and agarose removed by centrifugation through filter paper. DNA quality and quantity was checked with a Quant-iT dsDNA BR Assay kit (Invitrogen) on a Fluorescence Nanodrop.High throughput sequencing was performed at the Max Planck Institute for Evolutionary Biology (Ploen, Germany). Single-end, 150 bp reads were generated with a NextSeq 550 Output v2.5 kit (Illumina).