tools to determine a list of differentially expressed genes using
consensus from each of these methods. No matter which tool is
used, the key steps to determining which genes are differentially
expressed are normalization of raw expression count data and statistical comparison between phenotype groups. Each tool makes
different assumptions about the underlying distribution and variance of the expression data and uses different methods to normalize
and compare. Comparisons of these tools have been reported elsewhere [16–19].
Bioinformatics is an ever-evolving field, with new tools constantly developed and others replaced over time. Above, we have
outlined a workflow using commonly used tools that can be
accessed in their native forms or using Galaxy as a wrapper. The
full Galaxy Workflow can be accessed directly as well. It is our hope
that this chapter provides the reader with a foundation for
performing RNASeq experiments with mycobacteria.
The protocols below are specific to the parameters outlined
above for M. tuberculosis. All protocols and charts below have
been adapted from documentation developed by siTOOLs, KAPA
Biotechnologies, New England Biolabs (NEB), and Illumina.
2 Materials
2.1 Ribosomal RNA
Depletion
1. RNA sample: 50 ng to 1.5 μg, RNA integrity number
(RIN) > 7. For protocol to extract RNA from M. tuberculosis,
please see [20].
2. Ribopool rRNA depletion kit.
3. Hybridization buffer: 10 mM Tris–HCl (pH 7.5), 1 mM
EDTA, 2 M NaCl.
4. Depletion Buffer: 10 mM Tris–HCl (pH 7.5), 1 mM EDTA,
1 M NaCl.
5. Bead Resuspension Buffer: 0.1 M NaOH, 0.05 M NaCl.
6. Bead Wash Buffer: 0.1 M NaCl.
7. Thermocycler.
8. Magnetic bead rack.
9. 96-well PCR plate or strip tubes.
10. Nuclease-free water.
11. Dynabeads MyOne Streptavidin C1 (Thermo Fisher) (see
Note 2).
12. Agencourt RNAClean XP Beads.
13. 0.1Â TE buffer, pH 8.
14. 70% ethanol.
488
Shuyi Ma et al.
consensus from each of these methods. No matter which tool is
used, the key steps to determining which genes are differentially
expressed are normalization of raw expression count data and statistical comparison between phenotype groups. Each tool makes
different assumptions about the underlying distribution and variance of the expression data and uses different methods to normalize
and compare. Comparisons of these tools have been reported elsewhere [16–19].
Bioinformatics is an ever-evolving field, with new tools constantly developed and others replaced over time. Above, we have
outlined a workflow using commonly used tools that can be
accessed in their native forms or using Galaxy as a wrapper. The
full Galaxy Workflow can be accessed directly as well. It is our hope
that this chapter provides the reader with a foundation for
performing RNASeq experiments with mycobacteria.
The protocols below are specific to the parameters outlined
above for M. tuberculosis. All protocols and charts below have
been adapted from documentation developed by siTOOLs, KAPA
Biotechnologies, New England Biolabs (NEB), and Illumina.
2 Materials
2.1 Ribosomal RNA
Depletion
1. RNA sample: 50 ng to 1.5 μg, RNA integrity number
(RIN) > 7. For protocol to extract RNA from M. tuberculosis,
please see [20].
2. Ribopool rRNA depletion kit.
3. Hybridization buffer: 10 mM Tris–HCl (pH 7.5), 1 mM
EDTA, 2 M NaCl.
4. Depletion Buffer: 10 mM Tris–HCl (pH 7.5), 1 mM EDTA,
1 M NaCl.
5. Bead Resuspension Buffer: 0.1 M NaOH, 0.05 M NaCl.
6. Bead Wash Buffer: 0.1 M NaCl.
7. Thermocycler.
8. Magnetic bead rack.
9. 96-well PCR plate or strip tubes.
10. Nuclease-free water.
11. Dynabeads MyOne Streptavidin C1 (Thermo Fisher) (see
Note 2).
12. Agencourt RNAClean XP Beads.
13. 0.1Â TE buffer, pH 8.
14. 70% ethanol.
488
Shuyi Ma et al.