GSM4979308: RXR only R1; Salmonella enterica subsp. enterica serovar Typhimurium; RNA-Seq
Extraction of RNA was performed by initial incubation of the cells with TE buffer (pH 8.0) supplemented with 0.5 mg/ml lysozyme at RT for 5 min, with vortexing three times each 5 sec in between. Subsequently, the miRNeasy Mini kit was used and 3.5 volumes of RLT buffer was added, followed by addition of ethanol to adjust a final concentration of 60 %. After loading the sample volume, columns were washed according to the manual. The cDNA library preparation and sequencing was conducted at Vertis (Munich, Germany). The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' ends of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/µl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 75 bp read length.