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RNA-seq analysis of paired kidney-infiltrating and splenic T cells in the MRL/lpr murine model of systemic lupus erythematosus.

ABSTRACT: Purpose: The goal of this study is to compare the transcriptional phenotype of lymphoid and kidney-infiltrating T cell populations in the setting of systemic inflammatory disease to determine how tissue location alters their phenotype. Methods: mRNA profiles of T cells isolated from 23-week-old nephritic (protein score of 3+ on dipstick) mice were used in this study. T cells were isolated by flow cytometry gated on CD45+Thy1.1+CD44+ and either CD4 or CD8+ T cells. RNA was isolated using the RNeasy Plus Micro Kit (Qiagen). Samples were sequenced using Illumina NextSeq 500 with 75bp paired-end reads and aligned to the mm10 genome using the STAR aligner. The number of uniquely aligned reads ranged from 10 to 12 million. Using an optimized data analysis workflow, Gene-level counts were determined using featureCounts and raw counts were analyzed for differential expression using the “voom” method in the “limma” R package. Results: After determining genes that were differentially expressed between splenic T cells and KIT, we performed gene set enrichment analysis (GSEA. Differentially expressed genes were compared to several previously defined gene signatures that are characteristic of CD8+ and CD4+ T cell exhaustion in the chronic LCMV infection model and tumor infiltrating lymphocytes. Genes from the CD8+ exhaustion cluster were significantly enriched among genes that were differentially expressed in CD8+ KITs vs CD8+ splenocytes.

ORGANISM(S): Mus musculus

PROVIDER: GSE118271 | GEO | 2018/08/09

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study is to complare the transcitpional profiles of freshly isolated, splenic, murin GCBC and other activated and naive non-germinal center B cell populations. Methods: Cells were freshly isolated, bead purified and FACS Ari sorted . Sorted cells (1.5-3x106 cells per sample) were washed twice in cold PBS and RNA was prepared using shredder-columns (Qiagen; QIAshredder 79656) and the RNeasy Plus Mini kit (Qiagen) following the manufacturer’s instructions. 3 independent RNA libraries per cell population were prepared using Illumina TruSeq Stranded mRNA sample preparation kit. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by using dUTP in the Second Strand Marking Mix, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The products are then purified and enriched with PCR to create the final cDNA library. The cDNA libraries are validated using KAPA Biosystems primer premix kit with Illumina-compatible DNA primers and Qubit 2.0 fluorometer. Quality is examined using Agilent Tapestation 2200.The cDNA libraries were pooled at a final concentration 1.8pM. Samples were sequenced using Illumina NextSeq 500 with 75bp paired-end reads and aligned to the mm10 genome using the STAR aligner. The number of uniquely aligned reads ranged from 31 to 77 million. Using an optimized data analysis workflow, Gene-level counts were determined using featureCounts and raw counts were analyzed for differential expression using the “voom” method in the “limma” R package. Results: After determining genes that were differentially expressed between splenic GCBC and activated B cells, we performed gene set enrichment analysis (GSEA). Differentially expressed genes were compared to several previously defined hypoxic gene signatures.

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RNA-seq analysis of paired kidney-infiltrating and splenic T cells in the MRL/lpr murine model of systemic lupus erythematosus.

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