GSM8332117: sehA-OH-ara-positive; Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S; RNA-Seq
Total RNA was isolated using RNeasy Kit (Qiagen), according to the manufacturer's instructions. 5'-ends of hydroxylated or mono-phosphorylated RNA were identified using modified EMOTE protocol (Redder, 2015). 1 µg of total RNA was processed by Ribo-Zero Magnetic Kit (Bacteria). Resulting rRNA-depleted mRNA was phosphorylated by T4 polynucleotide kinase. Mono-phosphorylated transcripts was ligated to the synthesized RNA oligo Rp6. Ligated RNA was purified and reverse-transcribed using the DROAA primer, which contains sequencing barcode. Resulting cDNA was proceeded to PCR amplification using linker- (D6X) and DROAA-specific (B-PE-PCR20) primers. After 5 cycles, A-PE-PCR10 primer was added and continue 25 cycles to amplify PCR products.