GSM5605698: S. Typhimurium ∆metJ_SPI-1 Inducing Conditions_1; Salmonella enterica subsp. enterica serovar Typhimurium; RNA-Seq
RNAprotect Bacteria Reagent (Qiagen) in order to stabilize transcripts. After 5 minutes, bacteria were re-pelleted, and resuspended in 200mL of TE Buffer containing lysozyme (15mg/mL) and 20mL of Proteinase K. After addition of 700uL of buffer RLT+ bME, RNA was extracted using an RNeasy extraction kit (Qiagen). DNase was degraded with two Turbo DNase (Thermo-Fisher) per manufacturer instructions. To remove DNase after treatment, the solution was mixed with 350 L of -mercaptoethanol-containing RLT buffer, and then 700L of 96% ethanol was added. The mixture was then added to a RNeasy MinElute column (Qiagen) and RNA was reisolated according to manufacturer instructions. Illumina TruSeq Stranded total RNA-Seq Kit combined with Ribo-Zero rRNA removal kit (bacteria) was used to prepare total RNA-seq libraries. The rRNA depleted RNA was then reverse transcribed.  During the 2nd strand synthesis, the cDNA:RNA hybrid is converted into to double-stranded cDNA (dscDNA) and dUTP incorporated into the 2nd cDNA strand, effectively marking the second strand.  Illumina sequencing adapters were then ligated to the dscDNA fragments and amplified to produce the final RNA-seq library. The strand marked with dUTP is not amplified, allowing strand-specificity sequencing.  Libraries were indexed using a dual indexing approach allowing for multiple libraries to be pooled and sequenced on the same sequencing flow cell