GSM3003408: SDS treated sample beginning time point, replicate 1; Pseudomonas laurylsulfatovorans; RNA-Seq
Pellets from 5 ml of culture from each time point were resuspended in 1 ml of TE buffer and transferred to new tube with 250 μl of zirconia beads. One ml of TRIzol was added and each sample was mixed by vortexing for 3 minutes followed by adding 200 μl of chloroform and short mixing (30s vortex). After 30 minutes of incubation in RT the samples were centrifuged (10 minutes, 14000 rpm, 4°C). RNA was precipitated with isopropanol and sodium acetate for 30 minutes, pellet was centrifuged (20 minutes, 14000 rpm, 4°C), washed two times with 75% ethanol and resuspended in 40 μl of water. The concentration and purity of RNA solutions were determined by NanoDrop. The RNA quality was evaluated by agarose gel electrophoresis. Ten μg of RNA was treated with TURBO DNase (ThermoFisher) according to manufacturer protocol followed by phenol/chloroform extraction. The purified samples were stored in -80°C. RNA libraries were prepared using KAPA Stranded RNA-Seq Library Preparation Kit. 2,5 µg of total RNA was used as input. rRNA was removed using Ribo-Zero rRNA Removal Kit (Bacteria). Fragmentation and priming were done in 94°C for 8 minutes. The libraries were verified using a 2100 Bioanalyzer (Agilent) High-Sensitivity DNA Assay and KAPA Library Quantification Kit for the Illumina. Sequencing was performed using an Illumina NextSeq (2x75 bp).
GEO Accession
GSM3003408