title: GSM1940895: SmutAact_24h_1; Aggregatibacter actinomycetemcomitans; Streptococcus mutans; RNA-Seq
library_construction_protocol: For RNA isolation the pellets were resuspended in lysis buffer containing 10 mM Tris, 1 mM EDTA, pH 8.0, 2.5 mg/ml lysozyme (Sigma-Aldrich, Taufkirchen, Germany) and 50 U/ml mutanolysin (Sigma-Aldrich, Taufkirchen, Germany) and incubated at 25°C for 1.5 h at 350 rpm. Subsequently TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was added, samples were well mixed and incubated at room temperature for 5 min. Next the cells were vortexed for 10 times 30 s with 1 min intervals on ice in the presence of 50 µg sterile, acid-washed glass beads (diameter 106 µm; Sigma-Aldrich, Taufkirchen, Germany). Samples were briefly centrifuged and supernatants were vortexed with ice cooled 1-bromo-3-chloropropane (Sigma-Aldrich, Taufkirchen, Germany) for 15 s followed by 5 min incubation at room temperature. Next, samples were centrifuged (4°C, 15 min at 13,000 rpm) and RNA in collected aqueous phase was precipitated with 1 volume of ice cooled isopropanol and suspended in water. After removal of DNA with RNase free DNaseI (Qiagen, Hilden, Germany) using the in-solution digestion protocol, RNA samples were purified using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer`s instructions with the additional DNaseI on-column digestion. Bacterial rRNAs were depleted using MicrobExpress (Ambion, Austin, TX). mRNA was converted to cDNA using a ScriptSeq v2 RNA-Seq (Epicentre Biotechnologies, Madison, Wisconsin, USA).
identifiers.namespace: GEO
identifiers.id: GSM1940895