Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: To harvest the total RNA from host and pathogen at once, we treated cellular mixture with concentrated solution of ammonium sulfate to prevent any protein-dependent RNA degradation. Each ml of the ammonium sulfate solution (pH 5.2) contains 0.7 g (NH₄)₂SO₄, 20 mM EDTA and 25 mM Na-citrate. Ammonium sulfate solution is added directly to the medium with the ratio 3:1. The suspension was vigorously pipetted to ensure the complete mixing of ammonium sulfate and infection medium. Adherent host cells were scrapped and incubated further (room temperature, 5 min). The suspension is collected and centrifuged at full speed (20 min, 4°C). Supernatant was removed and cell pellet was snap-frozen with liquid nitrogen. The cell mixture contains host cells, adherent bacteria and non-adherent bacteria. With the aim of disrupting pneumococcal cells, a bead beating procedure was exercised. In a 1.5 ml screw cap tube, a PCR tube full of sterile, RNase free glass beads were added together with 50 μl 10% SDS and 500 μl phenol-chloroform. In the meantime, frozen cell pellet was resuspended in TE solution (10 mM Tris-HCl, 1 mM Na₂EDTA, pH 8.0, DEPC, diethylpyrocarbonate-treated solution). The cell suspension was added into the screw-capped tube and beat for 3 times 45s. Tubes were immediately placed in ice box and centrifuged at full speed, 4°C to separate organic and water phases. Water phase was pipetted out and back extraction were performed to the organic phase to optimize RNA yield. The subsequent part of the RNA isolation were performed based on High Pure RNA Isolation Kit (Roche) with column. Water phase from phenol-chloroform extraction were mixed well with binding buffer and pipetted into the upper chamber of column and centrifuged. DNase mix were then added onto the filter and incubated at room temperature for 30 min to digest total genomic DNA. Total RNA were eluted according to the manufacturer protocol. Total RNA samples were treated with a 1:1 mixture of Human/Mouse/Rat and Gram positive bacteria capture probes (Ribo-Zero rRNA Removal Kits, Illumina, US) to simultaneously deplete host and pathogen rRNA. cDNA library preparation was prepared with TruSeq® Stranded Total RNA Sample Preparation Kit (Illumina, US) according to the manufacturer protocol. RNA libraries were prepared for sequencing using standard Illumina protocols