title: Illumina HiSeq 1500 paired end sequencing; Dual RNA-seq of M. tuberculosis H37Rv infecting macrophage-like THP-1 cells during 48h
design: Dual RNA-seq of M. tuberculosis H37Rv infecting macrophage-like THP-1 cells during 48h
library_construction_protocol: The M. tuberculosis culture medium suspension was added to the differentiated cells and subsequently incubated for 4 h. The infection mix was removed, cells were washed twice with pre-warmed PBS and incubated further using fresh medium. RNA isolation and metabolite extraction was performed at designated time points p.i. Total RNA from mycobacterial cultures was prepared as previously described (Dietrich et al, 2000). Extraction of total RNA from THP-1 cells was prepared with TRIzol reagent using glycogen as a carrier according to the supplier’s recommendation (Life Technologies). Total RNA from M. tuberculosis-infected THP-1 cells without enrichment was isolated by abrasive particles in a reciprocal shaker with TRIzol (Dietrich et al, 2000). Enrichment of mycobacteria from infected THP-1 cells was carried out by differential lysis of host and mycobacterial cells using guanidine thiocyanate (GITC). The THP-1 cell line (American Type Culture Collection #TIB-202) was maintained in Roswell Park Memorial Institute medium 1640 supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate and 0.05 mM 2-mercapto-ethanol in a humidified 5% carbon dioxide atmosphere at 37°C. An estimated 5×106 cells/well in a 6-well plate were differentiated for 24 h using culture medium containing 40 ng/ml phorbol 12-myristate 13-acetate. Cells were then washed with fresh culture medium and incubated for 48 h. Exponentially grown M. tuberculosis H37Rv (American Type Culture Collection #27294) was pelleted (3,200 rpm, RT, 10 min), washed twice with phosphate-buffered saline and resuspended in THP-1 culture medium. THP-1 cells were infected at a multiplicity of infection (MOI) of 5. Barcoded RNA-seq libraries were prepared according to the TruSeq RNA Sample Preparation v2 Guide (Illumina) without fragmentation, as no additional fragmentation step was required because the average final library size was approximately 350 bp, and without size selection. The Gram-Positive Bacteria Ribo-Zero (Epicentre) rRNA Magnetic Removal Kit was used to remove bacterial rRNA from mycobacterial total RNA. For uninfected THP-1 total RNA at time point 0 h the Ribo-Zero Magnetic Kit Human/Mouse/Rat was used, while depletion of rRNA from infections without and with enrichment were depleted with the Ribo-Zero Magnetic Gold Kit Epidemiology. All cDNA libraries were verified for size distribution and quality using the high sensitive DNA kit (Agilent) on a 2100 Bioanalyzer and quantified with the Qubit 2.0 Fluorometer (Life Technologies).
attributes.tag: Experimental Factor: Mycobacterium tuberculosis H37Rv
attributes.value: organism
attributes.tag: Experimental Factor: grown in THP-1 cells
attributes.value: growth condition
attributes.tag: Experimental Factor: not applicable
attributes.value: infect
attributes.tag: Experimental Factor: 4
attributes.value: time