RNA-Seq uses next-gen sequencing to show the presence and quantity of RNA in a genome at a particular moment. DNASTAR’s SeqMan NGen application is the starting point for both reference-guided and de novo RNA-Seq workflows. Because this tutorial involves a reference-guided workflow, you will then use ArrayStar to analyze the completed RNA-Seq assembly.

In this tutorial, you will compare stationary phase RNA from wild-type E. coli cells with that from two different mutant cells, each lacking one of two genes that together encode a transcription factor for flagella and chemotaxis operons. An “operon” is a group of one or more genes that are transcribed as a single RNA unit.

In Part A, you will use SeqMan NGen to create a reference-guided DESeq2-normalized RNA-Seq assembly for two experimental replicates and one wild type control of E. coli transcription factors FlhC and FlhD. The data you will assemble is publicly-available single-end Illumina data from E. coli (Fitzgerald et al, 2014). In Parts B & C, you will use ArrayStar to perform downstream analysis and will learn to identify flagella-related operons using two different techniques.

IMPORTANT! There are two options for following this tutorial, one significantly longer than the other.

Item Full tutorial Abbreviated tutorial
Description Assemble in SeqMan NGen, then do downstream analysis in ArrayStar Do only the downstream analysis in ArrayStar, using a provided project
Download size 4.0 GB (unzips to 16 GB) 4.3 MB
Assembly wait time 1-3 hours none
Where to begin Perform the steps in Part A: Setting up the RNA-Seq reference-guided assembly in SeqMan NGen Read the steps in Part A: Setting up the RNA-Seq reference-guided assembly in SeqMan NGen without performing them

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