GSM5899064: M5_1 rep2; Pseudomonas fluorescens; ncRNA-Seq
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; doi:10.1093/nar/gkx005 ) and adapted for use in Pseudomonas fluorescens in (Ayan et al., 2020; doi:10.7554/eLife.57947).  Briefly, 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 24 sample-specific indices (Illumina) was added to each of the 24 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).