title: Ion Torrent Proton sequencing; RNA-seq of THP-1-derived macrophages infected with R. conorii and R. montanensis (1 hpi) against uninfected cells.
design: RNA-seq of THP-1-derived macrophages infected with R. conorii and R. montanensis (1 hpi) against uninfected cells.
library_name: Uninf4_s
library_construction_protocol: PMA-differentiated THP-1 cells monolayers were infected with R. conorii, R. montanensis at a multiplicity of infection of 10 or maintained uninfected and, at 1 hour post-infection, culture medium was removed and monolayers were washed 1x with PBS and scrapped. PMA-differentiated THP-1 cells monolayers at a cell confluency of 1.2 x 106 cells per well, in 6 well plates, were infected with R. conorii, R. montanensis at a multiplicity of infection of 10 or maintained uninfected. Plates were centrifuged at 300 g for 5 min at room temperature to induce contact between rickettsiae and host cells, and incubated at 34 °C and 5% CO2 for 1 hour. Total RNA was purified using SurePrep True Total RNA purification kit (ThermoFisher Scientific). DNA was removed from the RNA purification using Ambion Turbo DNAse. Removal of DNA contamination was verified by PCR. RNA was re-isolated using PureLink RNA Mini kit (Ambion).  After confirmation of RNA structural integrity, 5 μg RNA per sample were subjected to ribosomal RNA depletion using RiboMinusTM Eukaryote System v2 (Ambion) protocols. cDNA libraries were then constructed using the Ion Total RNAseq Kit v2 (Ion torrent).
attributes.tag: Experimental Factor: infect
attributes.value: mock