RNA Sequencing
From ActinoBase
- Extract and purify RNA samples according to RNA extraction from Actinobacteria. Add ERCC RNA Spike-In Mix if necessary.
- Check the quality of RNA samples using Agilent RNA 6000 Nano Kit or by running them on an agarose gel.
- Deplete rRNA using Illumina Ribo-Zero Plus rRNA Depletion Kit or a similar product
- Create Illumina RNA-seq libraries using TruSeq RNA Library Prep Kit.
- Sequence the libraries using an Illumina system such as NovaSeq 6000 Sequencing System.
- Trim low quality subsequences from reads using FASTX-Toolkit (single-end) or ngsShoRT (single- or paired-end).
- Align reads with the reference genome using HISAT2 or Bowtie2.
- Convert the SAM files into BAM files and sort and index the BAM files using Samtools.
- Count the number of reads overlapping with each feature such as CDS or tRNA gene using featureCounts or HTSeq.
- Perform a differential gene expression analysis using a Bioconductor package such as DESeq2.
