Single-Cell RNA-Seq Analysis of the Pineal Gland

Codebook for Mays et al., 2018 (PLOS ONE).

Study

Single-cell RNA sequencing of the mammalian pineal gland identifies two pinealocyte subtypes and cell type-specific daily patterns of gene expression

https://doi.org/10.1371/journal.pone.0205883

Mays JC, Kelly MC, Coon SL, Holtzclaw L, Rath MF, et al. (2018) Single-cell RNA sequencing of the mammalian pineal gland identifies two pinealocyte subtypes and cell type-specific daily patterns of gene expression. PLOS ONE 13(10): e0205883.

Running the Analysis Code

Single-Cell RNA-Seq analysis was performed using Seurat v2.2.0. Code should be run in the follow order:

Setup

SetupDependencies.R loads libraries, MetadataInit.R and GenerateMarkerReport.R load helper functions used in subsequent scripts.

./Scripts/SetupDependencies.R
./Scripts/MetadataInit.R
./Scripts/GenerateMarkerReport.R

Single Samples

These R Notebooks contain the analysis code for each individual sample.

./Day1.Rmd
./Day2.Rmd
./Night1.Rmd
./Night2.Rmd

Combined Samples

These R Notebooks contain the code that combines the treatment samples and applies a refined analysis.

./Day1n2_NoDoublets.Rmd
./Night1n2_NoDoublets.Rmd

Reference Genome Correction

The 3PrimeExtensionCorrection.R script applies corrected count values to the Seurat objects. Corrected count values were generated by re-aligning the sequencing data using a manually updated genomic reference. This does not alter PCA or TSNE embeddings, unless the PCA or TSNE functions are run again.

./Scripts/3PrimeExtensionCorrection.R

Figures

These scripts were used to generate the figures for the paper.

./Scripts/Figure_01.R
./Scripts/Figure_03.R
./Scripts/Figure_04.R
./Scripts/Figure_05.R