2.2 RNAseq Library
Preparation
1. NEBNext Ultra II RNA Library Prep Kit for Illumina.
2. Thermocycler.
3. Nuclease-free water.
4. Strip tubes or 96-well plate.
5. 80% ethanol.
6. 0.1Â TE buffer, pH 8.0.
7. Magnetic bead rack.
8. Agencourt AMPure XP beads.
2.3 Assess Library
Concentration
and Quality
1. KAPA Library Quantification Kits for Illumina.
2. 10 mM Tris–HCl, pH 8.0 + 0.05% Tween 20.
3. qPCR Thermocycler.
4. Qubit Bioanalyzer Assay Kit (with DNA High
Sensitivity Chip).
2.4 Pooling
Multiplexed Sample
Libraries
and Preparing
for Sequencing
1. Computer.
2. Illumina pooling calculator (https://support.illumina.com/
help/pooling-calculator/pooling-calculator.htm).
3. Illumina NextSeq 500 High Output Kit.
4. Freshly diluted 0.2 M NaOH.
5. 200 mM Tris–HCl, pH 7.
2.5 Sequencing
and Quality Control
of the Raw
Sequencing Data
1. Illumina NextSeq 500.
2. Illumina NextSeq 500 High Output Kit.
3. Computer (see Note 3).
4. FastQC software (available via Galaxy).
5. Trimmomatic software (available via Galaxy).
6. MultiQC software (available via Galaxy).
2.6 Alignment
to Reference Genome,
Reconstruction
of the Transcriptome,
and Differential
Expression Analysis
1. Computer (see Note 3).
2. Bowtie2 software (available via Galaxy).
3. M. tuberculosis whole genome (.FASTA format) (see Note 4).
4. htseq-count software (available via Galaxy).
5. featurecounts software (available via Galaxy).
6. M. tuberculosis genome annotation (.GTF format) (see Note 4).
7. DESeq2 software (available via Galaxy).
RNAseq Workflow
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