GSM8374379: S_L1; Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S; OTHER
The Salmonella were collected by centrifugation. Total RNA was extracted using RNA Extract Reagent (Evrogen, Russia) according to the manufacturer's protocol. DNA contaminants were removed using RNase-free DNase I kit (Ambion, USA). The integrity of the RNA was checked by Qsep100 bioanalyzer (BiOptic Inc.,Taiwan). Total RNA from Salmonella (5 μg) or Salmonella plus Acanthamoeba (10 μg) was capped with 3´ desthiobiotin-GTP using the Vaccinia Capping System (New England Biolabs, Ipswich, MA, USA). Capped RNA was then purified by Zymo Research Clean and Concentrator™-5 column using manufacturer instructions (Zymo Research, Irvine, CA, USA). Desthiobiotin-GTP-capped RNA was fragmented by polynucleotide kinase buffer and incubated for 5 min at 94 °C. The 3′-phosphates were removed from the fragmented RNA with polynucleotide kinase buffer and T4 polynucleotide kinase at 37 °C for 15 min. Streptavidin enrichment was performed by addition of capped RNA to 30 μL of prewashed hydrophilic streptavidin magnetic beads (New England Biolabs, Ipswich, MA, USA). The biotin-eluted RNA was collected by placing the tube on the magnetic rack. Eluted RNA was cleaned with AMPure XP beads (Beckman Coulter, Brea, CA, USA). Libraries were prepared from capped RNA using the NEBNext small RNA library prep kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer instructions. Library quality was assessed using a DNA high sensitivity chip on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) prior to sequencing.