essC_2hr_3
mixed sample
Protocols: 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
ENA first public
2026-01-01
INSDC center name
University of Warwick University of Cambridge
INSDC status
public
Submitter Id
E-MTAB-16250:essC_2hr_3
broker name
ArrayExpress
cell line
THP-1 macrophage
collection date
not collected
geographic location (country and/or sea)
not collected
isolate
not applicable
organism names
Staphylococcus aureus JE2 (NCBITaxon 381486) + Homo sapiens
sample name
E-MTAB-16250:essC_2hr_3
scientific_name
mixed sample
strain
ΔessC