This chapter focuses on RNAseq using NSR primers. This
protocol was used to identify QS-controlled genes in three related
Burkholderiaspecies,B. thailandensis,B. pseudomallei, andB. mal-
lei[4, 9],Pseudomonas aeruginosa[10], andRhodopseudomonas
palustris[11]. It is beyond the scope of this chapter to address
the details of data analysis. Simply put, large amounts of raw data
are generated for each sample following an RNAseq run. This data
must be analyzed for quality, sorted if barcodes were used, aligned
to a reference genome, and normalized prior to transcript analysis.
This can be done in a number of ways. It is best to consult with the
sequencing facility that will run the RNAseq samples for suggested
pipelines of data analysis. There are an increasing number of com-
mercially available software packages for NGS applications. When
choosing a software package, take note of the availability of webi-
nars, product manuals, and technical support services. These fea-
tures can be invaluable when processing data.2 Materials
2.1 RNA Preparation 1. RNAprotect Cell Reagent (Qiagen).
- Vortexer.
- Beadbeater for cell lysis (though not required if other methods
of cell lysis are used). - 0.1 mm silica beads (for use with beadbeater).
- 2 ml screw cap tubes with gasket (for use with beadbeater).
- Ice.
- Sterile, RNase-free tips.
- Tabletop microfuge.
- 1.5 and 2 ml microfuge tubes.
- miRNeasy Mini Kit (Qiagen).
- RNeasy MinElute Kit (Qiagen).
- 2-Mercaptoethanol (βME).
- Ethanol.
- NorthernMax-Gly Gel Prep/Running Buffer (Ambion, dilute
10 stock with DEPC treated RNase-free water). - 10TURBO DNase Buffer (Ambion).
- 2 Units (U)/μl TURBO DNase (Ambion).
- RNase-free water.
- Spectorphotometer for determining DNA and RNA
concentration. - RNAase AWAY Decontamination Reagent (ThermoFisher).
180 Charlotte D. Majerczyk