title: GSM4038656: N13-1507_a2; Listeria monocytogenes; RNA-Seq
library_construction_protocol: After one hour of acid exposure, or the respective control condition in BHI without acid, all reactions were stopped by the addition of 1 ml of 10 % acid phenol-chlorophorm in ethanol (v/v) to the cultures, which were chilled to 4 ¡C immediately. To extract the total RNA, the cultures were pelleted at 20Õ000 x g at 4 ¡C, resuspended in 200 ml of 10 mM Tris-buffer (Sigma-Aldrich, Buchs, Switzerland) containing 20 mg/ml Lysozyme (Sigma-Aldrich, Buchs, Switzerland) and 50 ml of 20 mg/ml Proteinase K (Qiagen, Venlo, the Netherlands) and subsequently lysed for 30 min at 37 ¡C. Cell lysates were then mixed with 1 ml TRI-Reagent (Invitrogen by Thermo Fischer Scientific, Zug, Switzerland) and bead-beaten for 2 x 60 s with cooling on ice in between in beat-beating tubes containing 1.4 mm ceramic beads (MagNA lyser green tubes, Roche, Rotkreuz, Switzerland). Subsequently the samples were centrifuged at 16Õ000 x g at 4 ¡C and the supernatant was transferred to a new tube, where 500 ml of bromo-chloro-propane (BCP, ACROS Organics, Geel, Belgium) were added. After 10 min incubation at room temperature the tubes were centrifuged for 15 min at 16Õ000 x g at 4 ¡C and the top aqueous layer was transferred into a new tube. Nucleic acids were precipitated overnight in 2.5 ml isopropanol at -80 ¡C. The following day, nucleic acids were pelleted, washed with 5 ml 75 % ethanol, resuspended in nuclease free water and adjusted to 500 ng/ml using a nanodrop (Witec AG, Sursee, Switzerland). 3 ml Turbo DNAse (Invitrogen by Thermo Fischer Scientific, Zug, Switzerland) and its buffer were added to digest the genomic DNA for one hour at 37 ¡C, with the addition of another 3 ml Turbo DNAse after 30 min to ensure complete digestion of genomic DNA. The DNAse treatment was followed by a phenol-chloroform (Fluka, Bucharest, Romania) extraction and RNA precipitation in EtOH with 1.6% (v/v) 3M sodium acetate at -80 ¡C overnight. The RNA was pelleted and washed with 70 % ethanol, dried and resuspended in 100 ml nuclease free water. Total RNA quality was assessed by a nanodrop measurement and an Agilent 2100 Bioanalyzer pico Chip. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Reagents (Bacteria)-Low Input and the Magnetic Core Kit-Low Input (Illumina, San Diego, USA) according to the manufacturerÕs specifications, and purified using the Agencourt RNAClean XP Kit (Beckman Coulter Inc., Brea, CA). A pico chip run on the bioanalyzer 2100 confirmed the depletion of 16S and 23S rRNA. Library preparation was done with an Illumina TruSeq RNA stranded kit according to the manufacturers instruction. All samples were pooled and sequenced on one lane of a Novaseq (paired-end, 150bp per read) at the Functional Genomics Centre ZŸrich.
attributes.tag: GEO Accession
attributes.value: GSM4038656