Overview

For this weeks tutorial we will use Kallisto to perform RNA-Seq pseudo-alignment to the reference cDNA (protein coding only). As with other weeks, containers have been prepared for you and a decription of the workflow via the command line.

1. Nextflow tutorial ~1 hour
2. DESeq2 Differential Expression Analysis in R ~ 1 hour

Please install the following packages in R Studio:

dplyr
biomaRt
tximport
rhdf5
gplots
DESeq2
apeglm
RColorBrewer
IHW
PCAtools
EnhancedVolcano
ComplexHeatmap
circlize
fgsea
tidyverse
ggpubr

Dataset

The researcher has captured exosomes from human malignant melanoma cell line (A375) and the human lung cancer cell line (A549) and added them to primary human epidermal melanocytes, (NHEM-c cells). There are triplicates of each cell line:

• NHEM-c (Control)
• NHEM-c + A549 exosomes (Lung)
• NHEM-c + A375 exosomes (Melanoma)

The researcher is interested in characterising the transcriptional profile of each transfected cell line, in an effort to elucidate the tumor pathways mediated by A375 & A549 tumor exosomes.