ScRNAseq-SMARTer-Analysis

This repository contains scripts used in the analysis of single-cell RNA-seq data generated using Smart-seq2 and SMARTer protocols from mouse thymocytes to study the order and logic of CD4 CD8 lineage choice and differentiation. The scripts in this repository are from the study -

Karimi, M. M., Guo, Y., Cui, X., Pallikonda, H. A., Horková, V., Wang, Y., . . . Merkenschlager, M. (2021). The order and logic of CD4 versus CD8 lineage choice and differentiation in mouse thymus. Nature Communications, 12(1). doi:10.1038/s41467-020-20306-w

• Alignment
• Quantification
• Analysis

Alignment

Smart-seq2 and SMARTer sequencing data were aligned with TopHat2 version 2.1.1 (Kim et al., 2013). GRCm38 (mm10) mouse genome reference was used for alignment and gene annotation from UCSC.

tophat2 \
	--transcriptome-index=</path/to/mm10_transcriptome_index/tophat2/genes> \
	--library-type=fr-firststrand \
	-p <num_of_threads> \
	-o </path/to/output/directory>/<sample_name> \
	</path/to/mm10_genome_index/Sequence/Bowtie2Index/genome> \
	</path/to/fastq/directory>/<sample_name>_R1.fastq.gz \
	</path/to/fastq/directory>/<sample_name>_R2.fastq.gz

Quantification

Read counts for genes were calculated using velocyto version 0.17 (La Manno et al., 2018) and the cell-gene count matrix obtained was used for downstream analysis.

velocyto run_smartseq2 \
	-d 1 \
	`find </path/to/aligned/bam/files> -name "*.bam" -print` \
	</path/to/mm10/genes.gtf> \
	-o </path/to/directory/velocyto-output>

Analysis

Requirements:

• R
• RStudio
• R packages:

     cluster
     cowplot
     DESeq2
     dplyr
     DT
     dynamicTreeCut
     dyno
     gam
     ggplot2
     ggpubr
     gridExtra
     jpeg
     kableExtra
     knitr
     magrittr
     Matrix
     metaMA
     org.Mm.eg.db
     RColorBrewer
     scater
     SCORPIUS
     scran
     Seurat
     slingshot
     splatter
     tidyverse
     tiff
     velocyto.R
     VennDiagram

Figure 1: PCA analysis of scRNA-seq data positions selection intermediates between pre-selection and post-selection thymocytes

a and b - Refer Figure1.Rmd and Figure1.html

Figure 2: scRNA-seq captures maturation, activation, and lineage identity as principal components of CD4/CD8 lineage choice and differentiation

a, b and c - Refer Figure2_Figure6ab_SupFigure2bcde.Rmd and Figure2_Figure6ab_SupFigure2bcde.html

Figure 4: Gene expression by individual selection of known Cd4 and Cd8a co-receptor gene expression status

a - Refer Figure4_SupFigure2a_SupFigure5.Rmd and Figure4_SupFigure2a_SupFigure5.html

Figure 5: Gene expression programs associated with distinct patterns of co-receptor gene activity in selection intermediates

a, c, d and f - Refer Figure5acdf_Figure6cd_SupFigure9b.Rmd and Figure5acdf_Figure6cd_SupFigure9b.html

b and e - Refer Figure5be_SupFigure9c_SupFigure11.Rmd and Figure5be_SupFigure9c_SupFigure11.html

Figure 6: CD4 and CD8 lineage characteristics are established at different times during CD4/CD8 lineage choice and differentiation

a and b - Refer Figure2_Figure6ab_SupFigure2bcde.Rmd and Figure2_Figure6ab_SupFigure2bcde.html

c and d - Refer Figure5acdf_Figure6cd_SupFigure9b.Rmd and Figure5acdf_Figure6cd_SupFigure9b.html

Figure 7: The temporal sequence of co-receptor gene expression by selection intermediates

a and b - Refer Figure7ab.Rmd and Figure7ab.html

e - Refer Figure7e.Rmd and Figure7e.html

g - Refer Figure7g.Rmd and Figure7g.html

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Identifying gene sets - F-set

Refer the R script - F-set_identification.R

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Supplementary Analysis and figures

Supplementary Figure 2: Key genes and correlations

a - Refer Figure4_SupFigure2a_SupFigure5.Rmd and Figure4_SupFigure2a_SupFigure5.html

b to e - Refer Figure2_Figure6ab_SupFigure2bcde.Rmd and Figure2_Figure6ab_SupFigure2bcde.html

Supplementary Figure 3: Co-expression of Zbtb7 and Runx3

a to e - Refer SupFigure3abcde_SupFigure8.Rmd and SupFigure3abcde_SupFigure8.html

e - Also refer SupFigure3e.Rmd and SupFigure3e.html

f - Refer SupFigure3f.Rmd and SupFigure3f.html

Supplementary Figure 5: Gene expression by individual selection intermediates in MHC class II-/- thymus.

a - Refer Figure4_SupFigure2a_SupFigure5.Rmd and Figure4_SupFigure2a_SupFigure5.html

Supplementary Figure 6: Details on gene expression programs associated with distinct patterns of co-receptor activity in selection intermediates

a to f - Refer SupFigure6.Rmd and SupFigure6.html

Supplementary Figure 8. Expression of activation versus lineage markers

a and b - Refer SupFigure3abcde_SupFigure8.Rmd and SupFigure3abcde_SupFigure8.html

Supplementary Figure 9. The temporal sequence of pre- and post-selection MHC class II-/- thymocytes and selection intermediates

b - Refer Figure5acdf_Figure6cd_SupFigure9b.Rmd and Figure5acdf_Figure6cd_SupFigure9b.html

c - Refer Figure5be_SupFigure9c_SupFigure11.Rmd and Figure5be_SupFigure9c_SupFigure11.html

Supplementary Figure 11. Cd4+ Cd8a- selection intermediates with NKT-like gene expression do not account for the timing of co-receptor gene expression patterns in the MHC class II-/- thymus

a and b - Refer Figure5be_SupFigure9c_SupFigure11.Rmd and Figure5be_SupFigure9c_SupFigure11.html

Also, refer SupFigure11.Rmd and SupFigure11.html

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