Today’s goal:

In this session, we will use the feature counts table generated by the star_salmon job to run a statistical analysis to identify differentially expressed genes.

Log into the HPC

To go to a shared directory for your project, for example: “kenna_team”. Type the following command and hit enter:

Go to the results of the previous Session 3:

Changing the folder permission for other members in the group to access the ‘results’ and ‘work’ folders:

Go to the folder with the feature counts generated by star_salmon:

Various ‘salmon.merged.*’ files are generated for all the samples processed in the experiment. Let’s look for the file that contains the feature counts for genes:

To browse the whole file type the following:

Copy the “salmon.merged.gene_counts.tsv” to your laptop and we will use it for statistical analysis using BioJupies (see below)

Differential gene expression analysis

Differential expression analysis using https://maayanlab.cloud/biojupies/analyze

From session 2, we will use the feature counts generated using the ‘star_salmon’ parameter setting. Either copy the relevant files (see below) to your laptop from the HPC or download the files below.

File	File name	File

File	File name	File
Gene feature counts	salmon_merged.gene_counts.tsv	Open   Open
Transcript feature counts	salmon_merged.transcript_counts.tsv	Open

Reading:

https://www.nature.com/articles/srep25533

Airway smooth muscle cells

Himes BE, Jiang X, Wagner P, Hu R, Wang Q, Klanderman B, Whitaker RM, Duan Q, Lasky-Su J, Nikolos C, Jester W, Johnson M, Panettieri R Jr, Tantisira KG, Weiss ST, Lu Q. “RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells.” PLoS One. 2014 Jun 13;9(6):e99625. PMID: 24926665. GEO: GSE52778.

From the abstract, a brief description of the RNA-Seq experiment on airway smooth muscle (ASM) cell lines: “Using RNA-Seq, a high-throughput sequencing method, we characterized transcriptomic changes in four primary human ASM cell lines that were treated with dexamethasone - a potent synthetic glucocorticoid (1 micromolar for 18 hours).”