GSM3724929: delta_lmo0599 replicate2; Listeria monocytogenes EGD-e; RNA-Seq
RNA extraction followed the protocol of Völker et al. 1995 (01 April 1994, Microbiology 140: 741-752, doi: 10.1099/00221287-140-4-741) with modifications. Cell pellets were resuspended in 1 ml Lysis Buffer I (25% sucrose; 20 mM Tris-HCl pH 8, 0.25 mM EDTA) and 2 µl of lysozyme (100 mg/ml) was added. After incubation for 5 min on ice, the samples were pelleted (15,000 x g for 30 s at 4°C) and resuspended in 300 µl Lysis Buffer II (3 mM EDTA, 200 mM NaCl). This solution was then mixed with 300 µl Lysis Buffer III (3 mM EDTA, 200 mM NaCl, 1% SDS) that had been pre-incubated at 95°C and the mixture was incubated at 95°C for five more minutes. 600 µl phenol/chloroform/isoamylalcohol (25:24:1) was added and vigorously mixed at room temperature for 5 min. Phases were separated by centrifugation (15,000 x g, 5 min), the aqueous upper phase was collected and the phenol/chloroform extraction was repeated. Afterwards, the aqueous phase was mixed with 600 µl chloroform/isoamylalcohol (25:1), shaken vigorously for 5 min and phases were separated by centrifugation (5 min at 15,000 x g). The chloroform extraction was repeated. Finally, RNA was precipitated by addition of 0.1 x volume 3 M sodium acetate (pH 5.2) and 1.5 volumes 96% ethanol and incubation at -20°C overnight and pelleted by centrifugation (15,000 x g for 15 min at 4°C). The pellet was washed with 70% ethanol and resuspended in 100 µl DEPC treated water. 10 µg total RNA were digested with DNAse using the RNase-Free DNase Set (Qiagen). RNA was then purified using RNA clean & concentrator columns (Zymo Research) for purification for RNA molecules longer than 200 nucleotides. RNA quality was assessed using Agilent Bioanalyzer RNA Nano chips. rRNA depletion was performed using the Ribo-Zero Bacteria Kit (Illumina) following the manufacturer's instructions. A total of approximately 2 µg purified RNA was trestedtreated with 10 µl Ribo-ZeroRemoval Solution and RNA concentrations were determined using a Qubit® fluorometer. After rRNA removal the remaining RNA was pelleted by ethanol precipitation following the recommendations in the Ribo-Zero protocol. RNA libraries were prepared using the TruSeq® Stranded mRNA Kit, starting with 19.5 µl Fragment, Prime, Finish Mix being added to the dried RNA pellet after rRNA depletion and ethanol precipitation. From this point on the manufacturers protocol was strictly followed (Illumina Part # 15031047 Rev. E). The RNA libraries were sequenced in paired-end mode with 2 times 76 cycles on the Illumina MiSeq (MiSeq® Reagent Kit v3; 150 cycles).
GEO
GSM3724929