Dual-transcriptome of Shigella flexneri infecting human HCT116 cells
Dual-transcriptome of Shigella flexneri infecting human HCT116 cells
Samples were washed with PBS and lysed directly in the dish with TRIzol.  HCT116 human colorectal cancer cells were plated 2 days before infection in 6-well plates for an approximate confluence of cells of 70% on the day of infection. Cells were infected with Shigella flexneri (strain M90T, MOI 100) previously grown into late exponential face (OD=0,7). Plates of cells with bacteria were centrifuged 10min at 300g and incubated 30min at 37°C, 5% CO2 and in humid atmosphere for invasion of cells by the bacteria. Extracellular bacteria were washed away by 3 PBS washes of the infected cells. Cells were then incubated in basal DMEM medium supplemented with 50µg/ml gentamicin for 5h in 37°C, 5% CO2 humid atmosphere.  Total RNA was purified by TRIzol (Life Technologies, 15596026) using 1ml of reagent for each 6 million of cells or less, according to the manufacturer's instructions. RNA samples were then subjected to DNase digestion (DNase TURBO, Life technologies), followed by acidic phenol/chloroform purification.  From the total RNA samples, ribosomal RNA molecules were depleted using the Ribo-Zero rRNA Removal Kit (Illumina) using a 1:1 ratio of probes from the Human/Mouse/Rat and Bacteria module.The rRNA depleted RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/µl using a high fidelity DNA polymerase (13 cycles). The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics).