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Introduction to RNA‐Seq Analysis

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Ayushi Agrawal edited this page Jun 23, 2026 · 2 revisions

Description

RNA-seq provides powerful insights into gene expression and cellular processes. Unlock the full potential of your RNA-seq data in this introductory, hands-on 3-session workshop covering a typical bulk RNA-seq workflow, from processing raw sequencing reads to identifying differentially expressed genes and interpreting their biological significance.

In the first two sessions, you'll learn how to perform a quality check of raw data in the formats provided by sequencing centers, trim adapters, map reads to a reference genome, and tally gene-wise counts. In session 3, you'll then bring those counts into R to model complex experimental designs and test hypotheses using edgeR's generalized linear models to extract real biological meaning from your data.

Learning Path

Intermediate   This is an intermediate-level workshop in the RNA-Seq Analysis series.

Prerequisites for demo: Participants should be comfortable running commands on the Unix command line and have prior experience with R.

Materials

Sessions 1 & 2

Session 3

Pre-Workshop Instructions

Please complete the following steps before the workshop.

Sessions 1 & 2

1. Install Docker Desktop

Docker allows you to run all bioinformatics tools without installing them individually. Everything runs inside a pre-built container.
Download: https://www.docker.com/products/docker-desktop/

macOS

  1. Download Docker Desktop for Mac
    • Choose Apple Silicon for M1/M2/M3/M4 Macs
    • Choose Intel for Intel Macs
    • Not sure? Click the Apple menu > About This Mac. Look for "Apple M1/M2/M3/M4" (Apple Silicon) or "Intel" (Intel).
  2. Open the .dmg file and drag Docker to Applications
  3. Launch Docker Desktop and wait for the whale icon in the menu bar to stop animating
  4. You do not need to log in or create a Docker account

Windows

  1. Download Docker Desktop for Windows
    • Most users: choose AMD64 (for Intel or AMD processors)
    • Surface Pro X or Snapdragon laptops: choose ARM64
    • Not sure? Choose AMD64 — it works on the vast majority of Windows computers
  2. Run the installer and follow the prompts
  3. Restart your computer when prompted
  4. If prompted for a WSL kernel update, open Command Prompt and run: 
    wsl --update
    Then restart Docker Desktop.
  5. Launch Docker Desktop. You do not need to log in or create a Docker account.

Linux

  1. Download and install Docker Desktop for Linux from the link above
  2. Launch Docker Desktop

2. Verify Docker Installation

Open your terminal:

  • Mac: Press Cmd + Space, type Terminal, press Enter
  • Windows: Press the Windows key, type cmd, press Enter
  • Linux: Press Ctrl + Alt + T

Run:

docker --version

You should see the Docker version printed. Then verify it can run containers:

docker run hello-world

If you see "Hello from Docker!", you are ready.

3. Download the Docker Image

In the same terminal, run:

docker pull nfcore/rnaseq

This downloads about 3 GB and may take several minutes. You only need to do this once. Verify the image was downloaded:

docker images

You should see nfcore/rnaseq in the list. This confirms the image was downloaded successfully.

4. Download the Workshop Materials

Download the hands-on materials folder from Dropbox.

Place the folder in an easy-to-find location:

OS Suggested location
Mac ~/Downloads/Intro_to_RNA-seq_data_analysis
Windows C:\Users\YourName\Downloads\Intro_to_RNA-seq_data_analysis
Linux ~/Downloads/Intro_to_RNA-seq_data_analysis

The folder contains:

  • DAY0_Setup_Instructions.pdf — Setup guide and Docker overview (this content)
  • DAY1_Quality_Control.pdf — Day 1 workshop guide
  • DAY2_Alignment_Quantification.pdf — Day 2 workshop guide
  • fastq/ — Raw sequencing data (4 FASTQ files)
  • reference_genome/ — Human chr16 reference (FASTA + GTF)

Note: You do not need to open or run the Day 1 or Day 2 guides before the workshop. We will walk through them together during the live sessions.

Session 3

Before the workshop, please make sure that you have R and RStudio installed on your laptops. Please also install the statmod, pheatmap, edgeR, tidyverse, magrittr, org.Mm.eg.db and ggplot2 packages in R. Follow this instructions:

  1. Open Rstudio
  2. Enter the commands below to install the required packages.
install.packages("magrittr")
install.packages("statmod")
install.packages("pheatmap") 
install.packages("BiocManager")
BiocManager::install("edgeR")
BiocManager::install("org.Mm.eg.db")
install.packages("tidyverse")
install.packages("ggplot2")
  1. Verify the correct installation uploading the library with library(package_name)
library(magrittr)
library(statmod)
library(pheatmap)
library(edgeR)
library(org.Mm.eg.db)
library(tidyverse)
library(ggplot2)

Additional preparation materials

  • During the workshop, we will focus on bulk RNA‑seq data analysis using fastq files as the starting point. We will not cover library preparation and sequencing. If you want background on these topics, please review this slide deck and this Illumina RNA‑seq playlist.
  • Familiarity with Unix is required for the demo. If you are not familiar with Unix, please watch the recording of our Intro to Unix workshop.

Troubleshooting

  • "Container name already in use" - Remove the old container using below command and your docker run command again. docker rm rnaseq_day1

  • Files missing inside container — Check that Docker Desktop is running and that the path in your -v flag is correct.

  • "Cannot connect to the Docker daemon" — Open Docker Desktop and wait for it to fully start (whale icon stops animating).

  • Docker is slow on Apple Silicon Macs — On M1/M2/M3/M4 Macs, Docker runs through an emulation layer. This is normal and expected. Steps like STAR indexing will take longer than on Intel systems.

  • Restarting a stopped container - If you exited a container but did not remove it, you can restart it:

    docker ps -a                 # List all containers
docker start -i rnaseq_day1  # Restart the container

Online Learning

You can access these materials remotely at any time and go through them at your own pace. Here's how:

  1. Download the materials here and unzip the contents.

  2. Inside the downloaded folder is a slide deck that contains notes below each slide where you can find links to recommended reading material.

  3. Each section contains relevant background on the tools/tech and describes files that should be used as input to the tools. Please go through these step-by-step and read the suggested reading material.

Additional materials after completion

Refer to our workshop materials for pathway analysis and materials from the UCSF library for bulk RNA-seq analysis, which focuses on differential gene expression analysis. The library hosts its materials on the UCSF Collaborative Learning Environment, which you can visit by following a link in the description of their materials.

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