Retina ganglion cells dataset

Maciej Daniszewski, Anne Senabouth, Duncan E. Crombie, Quan Nguyen, Samuel Lukowski, Tejal Kulkani, Donald J Zack, Alice Pébay, Joseph E. Powell and Alex W. Hewitt

Public repository for code associated with the processing of single cell retina ganglion cells, from the publication Single Cell RNA Sequencing of stem cell-derived retinal ganglion cells.

Raw Data

E-MTAB-6108 - Single cell RNA sequencing of stem cell-derived retinal ganglion cells

Required installations

Cell Ranger 1.3.1

Please refer to the 10x Genomics for information about this program.

R 3.4.2

Please refer to the R website for information about this program.

Instructions

Processing raw data with Cell Ranger 1.3.1

THIS STAGE SHOULD BE RUN ON A HIGH-PERFORMANCE COMPUTING ENVIRONMENT

1. Download the raw data from ArrayExpress.
2. Download the Cell Ranger 1.3.1 reference. You can also prepare your own by following the instructions here.
3. Edit project.xml to suit your computing environment. Make sure your paths do not end in '/'.
4. Run ProcessData.bash. If you are working on a cluster, you can call this from a cluster submission script. We have included one for PBSPro (SubmitPBS.pbs), which you can use as a template for other systems.

Processing Cell Ranger output with R

THIS STAGE CAN BE RUN ON A DESKTOP Run ProcessData.R, where the second argument points to the location of the files generated by Cell Ranger.

Rscript ProcessData.R RetinaGanglionCells

This will generate two clean expression matrices in the directory.

Exploring this dataset with 'ascend'

You can explore the clean data available on ArrayExpress by loading the expression matrices as follows:

# Load the expression matrices into R
sample1.matrix <- read.csv("iPSC_RGscRNASeq_Sample1.tsv", sep = "\t")
sample2.matrix <- read.csv("iPSC_RGscRNASeq_Sample2.tsv", sep = "\t")

# Connect the two matrices together using dplyr and set the rownames
library(dplyr)
expression.matrix <- full_join(sample1.matrix, sample2.matrix, by = "X")
rownames(expression.matrix) <- expression.matrix[ ,1]
expression.matrix <- expression.matrix[ ,2:ncol(expression.matrix)]

# Create an EMSet for use with ascend
em.set <- NewEMSet(ExpressionMatrix = expression.matrix)