6.2 Lab Activity: Single-Cell RNA-seq

6.2.1 Purpose

This lab will teach students how to explore single-cell RNA-seq data. This will enable them to use scRNA-seq data to investigate their scientific questions.

6.2.2 Learning Objectives

  1. Launch cellxgene on SciServer.
  2. Use cellxgene to determine which cell types express a gene.
  3. Compare expression of different genes across cell types.

6.2.3 Introduction

In this lab, students will explore single-cell RNA-seq data from the Drosophila gut, using data from the Fly Cell Atlas. The Fly Cell Atlas is a large collection of single-cell sequencing data from Drosophila (fruit flies), with the goal of creating a map of all the cell types in a fruit fly. Many scientists have contributed to the Fly Cell Atlas, and it is an incredible resource for anybody working with fruit flies.

Analyzing single-cell sequencing data is difficult, because there is just so much data! The cellxgene (pronounced “cell-by-gene”) tool provides an interactive visualization of the data, giving investigators a big-picture overview of the data and also enabling them to select specific cell types or genes for further investigation. The Fly Cell Atlas has made cellxgene available on its website (flycellatlas.org), but for this lab we will use cellxgene on SciServer as it offers more functionality (e.g. ability to identify differentially expressed genes). Learning how to use cellxgene will give students a valuable tool for investigating scientific questions.

Watch this video (video)(slides) to learn more about Fly Cell Atlas and cellxgene.

6.2.4 Activity 1 - Launch cellxgene on SciServer

Estimated time: 15 min

6.2.4.1 Instructions

  1. Log into SciServer.
  2. Follow the instructions in this video (video)(slides) to launch cellxgene on SciServer with the Drosophila gut data.
  3. Look at the data on SciServer and answer the question below.

6.2.4.2 Questions

How many cell types are annotated in the Drosophila gut dataset?

6.2.5 Activity 2 - Explore genes of interest identified by differential expression analyses

Estimated time: 15 min

6.2.5.1 Instructions

  1. Launch cellxgene on SciServer.
  2. Follow the instructions in this video (video) (slides) to learn how to explore the expression of genes of interest
  3. Set cellxgene to show the expression of the gene Cht8, and answer the question below. (You can refer back to the video for how to view expression of a specific gene.)

6.2.5.2 Questions

In which of the following cell types is the gene Cht8 expressed the most?

  • A) Crop
  • B) Cardia
  • C) Posterior midgut
  • D) Anterior midgut

6.2.6 Activity 3 - Differential expression and gene marker identification

Estimated time: 15 min

6.2.6.1 Instructions

  1. Launch cellxgene on SciServer.
  2. Follow the instructions in this video (video) (slides) to learn how to perform differential gene expression and explore genes of interest using cellxgene.

6.2.6.2 Questions

UMAP plot of single-cell RNA-seq data from the fruit fly gut. License: [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/)

“Gene markers” are genes that are expressed in one cell type and not in any others. We call them “markers” because we can use them to tell cell types apart.

Looking at the UMAP plots above, which gene (dpy or ect) is a better gene marker for the adult pylorus cells?


Explain your answer to the previous question.

6.2.7 Footnotes

6.2.7.1 Resources

6.2.7.2 Contributions and Affiliations

  • Javier Carpinteyro-Ponce Ph.D., Carnegie Institution for Science
  • Katherine Cox, Ph.D., Johns Hopkins University
  • Matthew McCoy, Ph.D., Stanford University
  • Frederick Tan, Ph.D., Carnegie Institution for Science

Last Revised: October 2023

All materials CC-BY unless noted otherwise.