title: Illumina HiSeq 3000 sequencing; RNAseq of Sinorhizobium meliloti 1021 treated with the chemically synthesized peptide DgDef1 delta SP delta CTPP (representing a truncated variant of the Defensin-like peptide DgDef1 from the actinorhizal plant Datisca glomerata) against an untreated control
design: RNAseq of Sinorhizobium meliloti 1021 treated with the chemically synthesized peptide DgDef1 delta SP delta CTPP (representing a truncated variant of the Defensin-like peptide DgDef1 from the actinorhizal plant Datisca glomerata) against an untreated control
library_construction_protocol: Rm1021 cells (OD600 = 0.5) were challenged in 10 mL TY medium with 25 g/mL of DgDef1 delta SP delta CTPP in 50 mL conical tubes at 30C, 150 rpm for 1h. The negative control was challenged with acetonitrile:water (1:3). Assays were performed in triplicates. Cells were centrifuged at 10.000 rpm for 5 min and supernatant was removed. Cell pellets were used for RNA isolation. For RNA isolation, cells pellets were resuspended in 1 mL of QIAzol lysis reagent and  homogenized in a FastPrep sample preparation system (3 x 6.500 rpm, 20 sec, 15 sec break) using Lysing Matrix B containing 0.1 mm silica beads (MP biomedicals, cat. no. 6911). Suspensions were then i) incubated 5 min at 15-25 C and 140 l of chloroform was added; ii) shaken vigorously for 15 sec and incubated for 3 min at 15-25 C, centrifuged (12.000 rpm, 15 min, 4C) to collect the upper aqueous phase, which was subsequently mixed with 1.5 volumes of absolute ethanol. The protocol proceeded with the miRNeasy Mini Kit (Qiagen, cat. no. 217004) according to the manufacturers instructions with an on-column DNase digestion.Since only a single library was prepared for each condition (i.e., treated and untreated), attention was paid to include RNA representing the four independent biological replicates in order to minimize the possibility of results biased by sample choice. Pooled samples used for RNAseq contained equal amounts of RNA from each biological replicate.  rRNA depletion was conducted with the Illumina Ribo-Zero rRNA removal Kit (Bacteria). The cDNA library was prepared using the NEB Ultra directional Kit