CD8 Follicular Analysis

Scripts for reproducing the RNA-seq analysis performed in the study: CD8 follicular T cells localize throughout the follicle during germinal center reactions and maintain cytolytic and helper properties.

You can find a free version of the article on PubMed.

PMID: 34274825

Data

Reference genome: GRCm38.p6

• RefSeq GCF_000001635.26

GEO accession: GSE177911

• CD8 Follicular Analysis
  • IL-2-KO CD4 Tfh & IL-2-KO CD8 Tfc

GEO accession: GSE112540

• Previously published IL-2-KO CD8 T cells

Alignment

## Building and index for the reference genome
# load the subread package
library(Rsubread)
ref <-("~/data/GCF_000001635.26_GRCm38.p6/GCF_000001635.26_GRCm38.p6_genomic.fna")
buildindex(basename="mus_reference_GRCm38_index",reference=ref)

## Alignment
align(index="~/data/GCF_000001635.26_GRCm38.p6/index/mus_reference_GRCm38_index",readfile1="~/data/follicular_fastqs/CD4_Tfh_1_R1.fastq",readfile2="~/data/follicular_fastqs/CD4_Tfh_1_R2.fastq", nBestLocations=10, output_file="~/data/follicular_fastqs/CD4_Tfh_1_alPEv3.BAM",nthreads=16,phredOffset=33)

align(index="~/data/GCF_000001635.26_GRCm38.p6/index/mus_reference_GRCm38_index",readfile1="~/data/follicular_fastqs/CD4_Tfh_2_R1.fastq",readfile2="~/data/follicular_fastqs/CD4_Tfh_2_R2.fastq", nBestLocations=10, output_file="~/data/follicular_fastqs/CD4_Tfh_2_alPEv3.BAM",nthreads=16,phredOffset=33)

align(index="~/data/GCF_000001635.26_GRCm38.p6/index/mus_reference_GRCm38_index",readfile1="~/data/follicular_fastqs/CD8_Tfc_1_R1.fastq",readfile2="~/data/follicular_fastqs/CD8_Tfc_1_R2.fastq", nBestLocations=10, output_file="~/data/follicular_fastqs/CD8_Tfc_1_alPEv3.BAM",nthreads=16,phredOffset=33)

align(index="~/data/GCF_000001635.26_GRCm38.p6/index/mus_reference_GRCm38_index",readfile1="~/data/follicular_fastqs/CD8_Tfc_2_R1.fastq",readfile2="~/data/follicular_fastqs/CD8_Tfc_2_R2.fastq", nBestLocations=10, output_file="~/data/follicular_fastqs/CD8_Tfc_2_alPEv3.BAM",nthreads=16,phredOffset=33)

## FeatureCounts
require(Rsubread)

gtf <- (file = "~/data/GCF_000001635.26_GRCm38.p6/test_GRCm38.p6_genomic.gtf")

setwd("~/data/follicular_fastqs/bam_v3")
bamdir <- ("~/data/follicular_fastqs/bam_v3")
bamfiles  <- dir(bamdir, pattern = ".BAM$")

# generate the counts matrices 
fc <- featureCounts(files=bamfiles, 
	GTF.featureType="gene",
	GTF.attrType="gene_id", 
	annot.ext=gtf, 
	isGTFAnnotationFile=TRUE, 
	countMultiMappingReads=TRUE, 
	fraction=TRUE, 
	isPairedEnd = TRUE, 
	countChimericFragments =FALSE, 
	nthreads = 8)


save.image("follicular_analysis_v3.RData")

Requirements

CLI Tools:

• fastqc

• trimmomatic

R Packages:

• featureCounts

• Rsubread

• limma

• edgeR

• clusterProfiler

• tidyverse

• extrafont

• gplots

• AnnotationDbi

• org.Mm.eg.db

Citation

Please cite if our repository was useful for your research:

@article{VALENTINE2021102690,
  author = {Kristen M. Valentine and Genevieve N. Mullins and Oscar A. Davalos and Lek Wei Seow and Katrina K. Hoyer},
  title = {CD8 follicular T cells localize throughout the follicle during germinal center reactions and maintain cytolytic and helper properties},
  journal = {Journal of Autoimmunity},
  volume = {123},
  pages = {102690},
  year = {2021},
  issn = {0896-8411},
  doi = {https://doi.org/10.1016/j.jaut.2021.102690},
  url = {https://www.sciencedirect.com/science/article/pii/S0896841121000986},
  keywords = {CD8 T follicular Cells, CXCR5+ CD8 T cell, Autoimmunity, Germinal center, T regulatory cell},
}