RNA Sequencing

Instructions

This procedure is an example and the optimal library preparation and data analysis procedures can be different depending on the type of the DNA sequencer used and purpose of the experiment.

1. Extract and purify RNA samples according to RNA extraction from Actinobacteria. Add ERCC RNA Spike-In Mix if necessary.
2. Check the quality of RNA samples using Agilent RNA 6000 Nano Kit or by running them on an agarose gel.
3. Deplete rRNA using Illumina Ribo-Zero Plus rRNA Depletion Kit or a similar product, or using the rRNA depletion with Streptomyces coelicolor A3(2) specific probes protocol.
4. Create Illumina RNA-seq libraries using TruSeq RNA Library Prep Kit.
5. Sequence the libraries using an Illumina system such as NovaSeq 6000 Sequencing System.
6. Trim low quality subsequences from reads using FASTX-Toolkit (single-end) or ngsShoRT (single- or paired-end).
7. Align reads with the reference genome using HISAT2 or Bowtie2.
8. Convert the SAM files into BAM files and sort and index the BAM files using Samtools.
9. Count the number of reads overlapping with each feature such as CDS or tRNA gene using featureCounts or HTSeq.
10. Perform a differential gene expression analysis using a Bioconductor package such as DESeq2.