Genome-wide identification of transcriptional start sites in the phytopathogen Dickeya dadantii 3937 based on differential RNA-Seq (dRNA-Seq)

ABSTRACT: In this study transcriptional start sites (TSS) for the phytopathogen Dickeya dadantii 3937 were determined based on differential RNA-seq (dRNA-seq) as previously described (Sharma and Vogel, 2014). RNAs from E-MTAB-541 (Jiang et al. 2015) were pooled into four samples S1 to S4 reflecting a variety of environmental conditions. Each of them resulted in a combination of stress conditions (pH, NaCl, H2O2) and growth conditions: exponential phase with stress (S1), exponential phase without stress (S2), stationary phase with stress (S3), stationary phase without stress (S4). Those samples were then supplied to Vertis Biotechnologie AG for TEX treatment and Illumina sequencing. Briefly, ribosomal RNA molecules were depleted from the total RNA samples using the Ribo-Zero rRNA Removal Kit for bacteria (Epicentre). From the rRNA depleted RNA samples, the small RNA fractions <200 nt were separated from the large fractions using the RNeasy MinElute Cleanup Kit (Qiagen). TSS cDNA libraries were generated from the rRNA depleted large RNA fractions. First, the samples were fragmented using RNase III. Samples were then poly(A)-tailed using poly(A) polymerase, and split into two halves. One half was treated with Terminator exonuclease (+TEX, Epicentre), while the other one was left untreated (-TEX). 5'PPP structures were then converted into 5'P ends using RNA 5' Polyphosphatase (5'PP, Epicentre). Afterwards, an RNA adapter was ligated to the 5'P of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified using a high fidelity DNA polymerase, and purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). The cDNA pools were then sequenced on an Illumina NextSeq 500 system using 60 bp read length and a single-end, strand-specific protocol. Sequencing reads were trimmed, and both polyA and adapters removed.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Dickeya dadantii

SUBMITTER: Raphaël Forquet 

PROVIDER: E-MTAB-9075 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress