DOI: 10.5061/dryad.5qfttdzdf
title: RNAseq of tolerized auroreactive RBC-specific CD4 T cells, HODxOTII murine model of autoimmune hemolytic anemia (AIHA)

description: We have previously identified that T cell tolerance is the stopgap to red
 blood cell (RBC)-specific autoimmunity. We have also shown that while
 tolerance is robust when mice are young, it fails upon age and leads to
 development of RBC autoantibodies. Herein, we set out to determine which
 transcripts were associated with tolerization of RBC-autoreative CD4 T
 cells. For these studies, we used a murine model of autoimmune hemolytic
 anemia (AIHA), whereby HOD mice (expressing a known RBC-specific antigen)
 were bred with OTII animals (which react to the ovalbumin epitope
 contained within teh HOD antigen) [see PMCID PMC8634489]. To that end,
 splenocytes were collected from 10-12 week old HODxOTII F1 mice (n=4 each
 genotype) and stained with antibodies against Thy1.2, Ter119, CD19, and
 CD4. CD19+ B cells and Ter119+ RBCs cells were excluded from live singlets
 and CD4+Thy1.2+ T cells were sorted. Total RNA was harvested using RNeasy
 mini plus kit (Qiagen). Samples were sent to the Genome Technology Access
 Center in the Department of Genetics at Washington University School of
 Medicine for library preparation and RNAseq. Each sample was run on 3
 lanes, with paired-end reads, and 2x150nts. 
description: Samples were prepared according to library kit
 manufacturer's protocol, indexed, pooled, and sequenced on an
 Illumina HiSeq. Data analysis was performed by TAC Genomics
 (https://tacgenomics.com). The reads were first mapped to the latest UCSC
 transcript set using Bowtie2 version 2.1.0 and the gene expression level
 was estimaed using RSEM v1.2.15. Differentially expressed genes were
 identified using the edgeR program. Genes showing altered expression with
 p &lt; 0.05 and more than 1.5 fold changes were considered
 differentially expressed. Goseq was used to perform the GO enrichment
 analysis and Kobas was used to perform the pathway analysis.
description: # HODxOTII CD4 T cell RNAseq
 [https://doi.org/10.5061/dryad.5qfttdzdf](https://doi.org/10.5061/dryad.5qfttdzdf) CD4+ T cells were sorted from HODxOTII F1 mice on a C57Bl/6J background. HOD is a model antigen containing hen egg lysosome, ovalbumin, and human blood group molecule Duffy. HOD is expressed in a red blood cell (RBC)-specific fashion. CD4 OTII T cells react to an epitope within ovalbumin. Thus, HOD+OTII+ mice have RBC autoreactive CD4 T cells. For these studies, CD4 T cells were sorted from autoreactive HOD+OTII+ and littermate HOD-OTII+ negative controls. The goal of the RNAseq was to determine which genes were associated with CD4 T cells tolerance to RBC-specific autoantigens. There are 8 total samples: HOD+OTII+ (n=4) and HOD-OTII+ (n=4). Each sample was run in 3 lanes, with paired-end reads. As such, there are 6 Fastq files (where 5_1/5_4, 6_1/6_4, 7_1/7_4 are paired reads). Each raw data file has been uploaded with the genotype and mouse number in the file name.  Individual fq.gz files are uploaded. **For autoreactive, tolerized, CD4+ T cells** **HOD+OTII+ Mouse #1** * Paired read #1 run_2895_s_5_1_withindex_HODposOTIIpos__1.fq.gz run_2895_s_5_4_withindex_HODposOTIIpos__1.fq.gz *  Paired read #2 run_2895_s_6_1_withindex_HODposOTIIpos__1.fq.gz run_2895_s_6_4_withindex_HODposOTIIpos__1.fq.gz *  Paired read #3 run_2895_s_7_1_withindex_HODposOTIIpos__1.fq.gz run_2895_s_7_4_withindex_HODposOTIIpos__1.fq.gz **HOD+OTII+ Mouse #2** * Paired read #1 run_2895_s_5_1_withindex_HODposOTIIpos__2.fq.gz run_2895_s_5_4_withindex_HODposOTIIpos__2.fq.gz *  Paired read #2 run_2895_s_6_1_withindex_HODposOTIIpos__2.fq.gz run_2895_s_6_4_withindex_HODposOTIIpos__2.fq.gz *  Paired read #3 run_2895_s_7_1_withindex_HODposOTIIpos__2.fq.gz run_2895_s_7_4_withindex_HODposOTIIpos__2.fq.gz **HOD+OTII+ Mouse #3** * Paired read #1 run_2895_s_5_1_withindex_HODposOTIIpos__3.fq.gz run_2895_s_5_4_withindex_HODposOTIIpos__3.fq.gz * Paired read #2 run_2895_s_6_1_withindex_HODposOTIIpos__3.fq.gz run_2895_s_6_4_withindex_HODposOTIIpos__3.fq.gz * Paired read #3 run_2895_s_7_1_withindex_HODposOTIIpos__3.fq.gz run_2895_s_7_4_withindex_HODposOTIIpos__3.fq.gz **HOD+OTII+ Mouse #4** * Paired read #1 run_2895_s_5_1_withindex_HODposOTIIpos__4.fq.gz run_2895_s_5_4_withindex_HODposOTIIpos__4.fq.gz * Paired read #2 run_2895_s_6_1_withindex_HODposOTIIpos__4.fq.gz run_2895_s_6_4_withindex_HODposOTIIpos__4.fq.gz * Paired read #3 run_2895_s_7_1_withindex_HODposOTIIpos__4.fq.gz run_2895_s_7_4_withindex_HODposOTIIpos__4.fq.gz **For littermate negative control CD4+ T cells** **HOD-OTII+ Mouse #1** * Paired read #1 run_2895_s_5_1_withindex_HODnegOTIIpos__1.fq.gz run_2895_s_5_4_withindex_HODnegOTIIpos__1.fq.gz * Paired read #2 run_2895_s_6_1_withindex_HODnegOTIIpos__1.fq.gz run_2895_s_6_4_withindex_HODnegOTIIpos__1.fq.gz * Paired read #3 run_2895_s_7_1_withindex_HODnegOTIIpos__1.fq.gz run_2895_s_7_4_withindex_HODnegOTIIpos__1.fq.gz **HOD-OTII+ Mouse #2** * Paired read #1 run_2895_s_5_1_withindex_HODnegOTIIpos__2.fq.gz run_2895_s_5_4_withindex_HODnegOTIIpos__2.fq.gz * Paired read #2 run_2895_s_6_1_withindex_HODnegOTIIpos__2.fq.gz run_2895_s_6_4_withindex_HODnegOTIIpos__2.fq.gz * Paired read #3 run_2895_s_7_1_withindex_HODnegOTIIpos__2.fq.gz run_2895_s_7_4_withindex_HODnegOTIIpos__2.fq.gz **HOD-OTII+ Mouse #3** * Paired read #1 run_2895_s_5_1_withindex_HODnegOTIIpos__3.fq.gz run_2895_s_5_4_withindex_HODnegOTIIpos__3.fq.gz * Paired read #2 run_2895_s_6_1_withindex_HODnegOTIIpos__3.fq.gz run_2895_s_6_4_withindex_HODnegOTIIpos__3.fq.gz * Paired read #3 run_2895_s_7_1_withindex_HODnegOTIIpos__3.fq.gz run_2895_s_7_4_withindex_HODnegOTIIpos__3.fq.gz **HOD-OTII+ Mouse #4** * Paired read #1 run_2895_s_5_1_withindex_HODnegOTIIpos__4.fq.gz run_2895_s_5_4_withindex_HODnegOTIIpos__4.fq.gz * Paired read #2 run_2895_s_6_1_withindex_HODnegOTIIpos__4.fq.gz run_2895_s_6_4_withindex_HODnegOTIIpos__4.fq.gz * Paired read #3 run_2895_s_7_1_withindex_HODnegOTIIpos__4.fq.gz run_2895_s_7_4_withindex_HODnegOTIIpos__4.fq.gz Three excel files are also provided. * This file contains the normalized read counts per million: HODposOTIIpos_v_HODnegOTIIpos_CD4_T_cells_TMM_normalized_reads_count_per_million.xls * This file contains the analyzed fold change, p-values, and false discover rate of all genes/transcripts, comparing HOD+OTII+ and HOD-OTII+ CD4 T cells: HODposOTIIpos_v_HODnegOTIIpos_CD4T_cells_all_detectable_genes.xls *  GO Pathway analysis between HOD+OTII+ and HOD-OTII+ CD4+ T cells HODposOTIIpos_vs_HODnegOTIIpos_GO_enrichment.xls Missing data code: NA