Preparing DESeq2 - ccsstudentmentors/tutorials GitHub Wiki

Step 1: Download the files produced by HTSeq-count for each of your samples (this was also the last step of the previous instruction file). Put these files in a working directory on your computer that will be convenient to work in. I am just going to call it: C:\RNASeq\ReadCounts\ .

Go ahead and make additional folders there for our eventual output:

C:\RNASeq\DESeq2Output\
C:\RNASeq\DESeq2DEGenes\

For this analysis, we will use a tool that is part of the Bioconductor package in R, which is a free software toolkit for statistical analyses. Step 2: Download and install R: https://cran.r-project.org/bin/windows/base/
Step 3: Donwload and install R-Studio: https://www.rstudio.com/products/rstudio/download/
Step 4: Launch R-Studio. Move to your RNA-Seq directory with the following command:

setwd('C:/RNASeq/')

Note: no, that isn't a typo, R uses forward slashes to separate folders even in Windows

Step 5: Install biocLite (the installation/updating software for bioconductor):

source('http://bioconductor.org/biocLite.R')

If you are asked to update all, some, or none of the dependent packages, type 'a' and then hit the 'enter' key to update all of them

Step 6: Install DESeq2

biocLite('DESeq2')

If you are asked to update all, some, or none of the dependent packages, type 'a' and then hit the 'enter' key to update all of them

Step 7: Load DESeq2

library(DESeq2)

Proceed to the section on simple DESeq2 data analysis.
If you used gene names instead of Ensembl IDs when counting reads, head to this section instead.

If you performed a complex RNA-Seq experiment with more than one experimental group and one control group, read through the simple analysis section first, then head to the section on multi-variate analysis with DESeq2.