Dickeya dadantii small RNA sequencing
Dickeya dadantii 3937 was grown at 30C in minimal M63 liquid medium supplemented with 0.2% (wt/vol) sucrose as the carbon source and 0.2% (wt/vol) polygalacturonate. Cells were grown until the exponential or early stationary phases (OD600 0.2 and 1.5 respectively). Aliquots were then transferred to four flasks, one of them was kept as a control and the other three were subjected to different stresses as previously described (REF). Briefly, acidic stress was induced by a 15 min incubation in the presence of 30 mM malic acid, resulting in a final pH of 5.0; (ii) oxidative stress was induced by a 15 min incubation in the presence of 100 M H2O2; (iii) osmotic stress was induced by a 15 min incubation in the presence of 300 mM NaCl. Total RNA from each D. dadantii sample was isolated using the frozen acid phenol method (Maes et Messens 1992), treated with DNase I and then submitted to Vertis Biotechnologie AG (http://www.vertis-biotech.com/) for commercial RNA-Seq. Briefly, RNA samples were pooled in equimolar amounts before the separation of the small RNA fraction (< 500 nt) from the large using the RNeasy MinElut Cleanup kit (Qiagen) and the rRNA depletion using the Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina/Epicentre). One half of the RNA was further treated with Terminator exonuclease (+TEX) and the other half was left untreated (-TEX). Then, the 5'PPP structures were removed from both samples using tobacco acid pyrophosphatase (TAP; Epicentre). Oligonucleotide adapters were ligated to the 5' and 3' ends of the RNA samples, allowing strand-specific sequencing. First-strand cDNA synthesis was performed using MMLV reverse transcriptase and the resulting cDNAs were amplified with PCR (15 cycles) using a high fidelity DNA polymerase. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics).
SUB4501091
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