GSM4721839: Flight Infected 1-2; Homo sapiens; Salmonella enterica subsp. enterica serovar Typhimurium; RNA-Seq
RNA was extracted from duplicate samples using the miRNeasy kit (Qiagen).  All samples were analyzed in biological duplicate (two separate bioreactors), except for the infected spaceflight sample which was analyzed in technical duplicate from the same bioreactor. Samples were lysed with 700 µl Qiazol (Qiagen), vortexed extensively and passed several times through a 25G needle. After a 5-minute static incubation at room temperature with intermittent vortexing, samples were added to MaXtract High Density tubes (Qiagen) together with 100 µl RNase-free water (Qiagen) and 200 µl chloroform (Sigma-Aldrich). Samples were then processed (including on-column DNase-treatment) according to the manufacturer's instructions. RNA was quantified using a Nanodrop spectrophotometer and the integrity validated using an Agilent Bioanalyzer 2100 to confirm a RIN of 7 or greater for all samples. One of the uninfected ground control samples (CN122) had a RIN of 6.4. RNA was fragmented with heat and magnesium to roughly 300 bp using the KAPA HyperPrep RNA-seq kit (Roche). The KAPA Hyper RNA-seq kit and Illumina-compatible adapters (IDT) were used for the remaining library construction. Adapter ligated molecules were cleaned using Kapa pure beads (Kapa Biosciences), amplified with Kapa HIFI enzyme. Each library was then analyzed for fragment size on an Agilent's Tapestation and quantified by qPCR (KAPA Library Quantification Kit) on Quantstudio 5 (Thermo Fisher Scientific). Libraries were then multiplexed and sequenced on 2x150 flow cell on the NovaSeq platform (Illumina) at University of Colorado Anschutz Medical Campus Genomics and Microarray Core.
GEO Accession
GSM4721839