title: GSM6917546: 9-4_KO_Ctrl; Homo sapiens; RNA-Seq
library_construction_protocol: After sorting, RNA was immediately isolated from collected cells using mirVana miRNA Isolation Kit's total RNA protocol (ThermoFisher #AM1560). Prior to extraction, samples in RNAlater were diluted 3x with PBS, spun down at 5,000 xg, and aspirated to remove RNAlater. After resuspending cells in the kit's L/B buffer, samples were vortexed for 60 sec to ensure lysis of Salmonella. Following total RNA extraction, gDNA was removed with 4 U TURBO DNase (ThermoFisher #AM2238) per μg of RNA and then purified with RNeasy MinElute cleanup kit (Qiagen #74204). In the gDNA-free RNA, the relative human to bacterial mRNA ratio was determined by RT-qPCR measurement of human ACTB and S. Typhi rpoD. In brief, 150ng of RNA was reverse transcribed with iScript cDNA synthesis kit (Bio-Rad #1708891) and 1:5 dilution of this cDNA was analyzed using iTaq Universal SYBR Green Supermix (Bio-Rad #1725124) and primers listed in Table S3 on a QuantStudio 3 System (ThermoFisher). This ratio was used to combine uninfected THP-1 mRNA and late-log S. Typhi inoculum mRNA for the control samples following Westermann & Vogel's approach mRNA was isolated and sequenced at Duke Sequencing and Genomic Technologies (SGT). cDNA library with depleted rRNA was followed with Illumina Standard Total RNA Prep using Ribo-Zero Plus (Illumina #20037135).