GSM2299965: GH_P_241_15_GATCAG_L005; Salmonella enterica subsp. enterica serovar Typhimurium; RNA-Seq
RNA was isolated from  Salmonella cells from swarm edges, 7 h from the time cells were spotted on swarm agar plates. Cells were suspended in RNA stabilization reagent [J. Microbiol. Methods 55, 399-409 (2003)] and stored at -70 0 C till proceeding for RNA isolation.  Cells were suspended in hot TRIzol RNA isolation reagent (LifeTechnologies, Carlsbad, CA) and processed for RNA isolation as described [Microbiol Discov. 2013; 1:10. http://dx.doi.org/10.7243/2052-6180-1-10]. RNA was treated with DNase three times (twice on column and once in solution) and DNA contamination was checked by PCR using primers against rpoD and 16S RNA genes. The messenger RNA population from all RNA samples was enriched by depleting rRNA sequences and strand-specific libraries were constructed as described earlier [Adv Microbiol 4, 25-32 (2014); Arch Microbiol 198, 353-362 (2016)].  Briefly, starting with 8 - 50 ng rRNA-depleted RNA, random-primed cDNA synthesis was done using ScriptSeq v2 RNA-Seq library preparation kit (Epicenter, WI).  cDNA was purified using Agencourt AMPure XP system (BeckmanCoulter, NJ). Libraries were amplified using FailSafe PCR enzyme kit (Epicenter, WI). Typically 12 (50-100 ng starting RNA) or 15 (8 ng starting RNA) PCR cycles were used and reverse primer from the kit was replaced with one of the ScriptSeq Index primers.  After PCR amplification, libraries were purified and size selected (~280 bp) using the Agencourt AMPure XP system (BeckmanCoulter, NJ). Profiles of library insert sizes were verified on the Experion microfluidic platform using 1K DNA chip (Bio-Rad Laboratories, CA).