Dual RNA-seq of THP-1 macrophages infected with JE2 WT and ΔessC at 2 and 6 hours post infection
Dual RNA-seq of THP-1 macrophages infected with JE2 WT and ΔessC at 2 and 6 hours post infection
THP-1 macrophages were infected with S. aureus JE2 WT or ΔessC strains in 12 well tissue culture treated plates for 1 hour before extracellular killing of bacteria. At both 2 and 6 hours post infection, wells were washed with PBS, before addition of LETs buffer (0.1M LiCl, 0.01 M Na2EDTA, 0.01 M Tris-Cl pH 7.4, 0.2% SDS) to the wells, and immediately frozen in dry ice.  Total RNA was extracted from samples by bead beating with lysing matrix B beads of 0.1 mm (MP Biomedicals) in a FastPrep-24  5G bead beater (MP Biomedicals) at 6.5 m/s for 20 seconds and rest in ice for 3 minutes, carried out for 6 cycles. RNA was extracted by addition of TRIzol Reagent and chloroform following manufacturer's instructions. RNA was precipitated with isopropanol, centrifuged, and then washed with 75% ethanol and finally resuspended in DNase- and RNase-free water. DNA and RNA were quantified by Qubit Broad Range assays (Invitrogen). Contaminating DNA was removed using TURBO DNA-free Kit (Invitrogen) following instructions for rigorous treatment. Samples were further cleaned up with LiCl Precipitation Solution 7.5 M (Invitrogen). RNA integrity was assessed by Agilent 6000 RNA Pico bioanalyzer, and RNA purity and contaminants checked by NanoDrop One (ThermoFisher).  Library construction was carried out by Genewiz Azenta. rRNA depletion was performed using NEBnext rRNA Depeletion Kit (Human/Mouse/Rat and/or bacteria). RNA sequencing library preparation was performed using NEBNext Ultra II RNA Library Prep Kit for Illumina by following manufacturer's instruction. First strand and second strand cDNA were subsequently synthesised. cDNA fragments were end-repaired and adenylated at 3'ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using NGS Kit