Browsing by Author "Ulrich, Benjamin"

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    Alternative Splicing Profile Comparison of Differentiating I-helper Cells to Dissect the Splicing Signatures of Th1, Th2, 1h17 and Treg Cells
    Lakshmipati, Deepak Kumar; Quoseena, Mir; Ulrich, Benjamin; Kaplan, Mark; Janga, Sarath Chandra
    This study focuses on the contribution of Alternative Splicing (AS) events in the differentiation and post-differentiation functions of T-helper cells, specifically in Thl, Th2, Th9, Th17 and Treg cells. T cell RNA-seq data from 72hr and 2week post differentiation time points was analyzed using (r-MATS) for alternative splicing events. We observed majority of the significant events are Skipped Exon (SE) events originating from a total of 1,556 genes and lntron Retention (RI) events were the second most abundant event occurring in 1,254 genes at 72 hours post differentiation. These numbers were significantly lower at 2 weeks post differentiation. PCR and qPCR validations confirmed scores of novel splicing event predictions. Results showed several skipped exon (SE) events in KTNl, IL4RA IL27, Hnrmpd, CREM and Arid4b showing different mRNA isoforms across multiple naïve vs differentiated T cell combinations. Overall, RI event associated genes were more prevalent (3,239 genes) than those exhibiting SE (2810 genes). SE events were associated with 10.8% (Th17), 11.2% (Treg), 12.1% (Th2) and 13.9% (Thl) of the genes, a similar trend was observed with RI events with a prevalence of 12.2% (Th17), 12.5% (Treg), 14.2% (Thl) and 14.4% (Th2) of the genes. Gene ontology results showed most of the genes showing SE and RI events are involved in processes like 'mRNA Processing', 'RNA Processing' and 'RNA Binding' and ontology results for retained introns also showed p53 suppression proteins, regulated exocytosis of neurotransmitters and hormones. It was also observed that Introns consistently favored retention at the 3' end of the gene than the 5', with 430 genes showing intron retention events at the 3' end and 21 genes exhibiting them at the 5' end, for the 72 hour time point. Enriched functional ontologies were consistently seen across all cell types to be exclusive for the genes showing RI in the 5' end vs the 3' end.
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    Identification of Splice Isoforms in T Helper (TH) Cells Using Nanopore-based cDNA sequencing
    (2020) Mir, Quoseena; Srivastava, Rajneesh; Ulrich, Benjamin; Kaplan, Mark H.; Janga, Sarath Chandra
    Naïve T cells are the precursors for the adaptive immune response against pathogens. They circulate the blood and secondary lymphoid organs until they are stimulated through a diverse pool of T cell receptors to become effector or memory cells. The differentiation of naïve T cells to effector subsets is regulated through a complex gene regulatory network resulting in transcriptomic alterations. Our understanding of how this process is controlled transcriptionally is limited. Nanopore-based RNA sequencing technology enables identification of the full length transcripts and alternatively spliced isoforms. We used this technique to identify the transcriptional signatures of differentiated T cell subtypes. Here, we present a single molecule transcriptome map of differentiated Treg cells obtained from mouse spleen to characterize full-length transcripts and alternatively spliced isoforms. We obtained 5,092,757 quality filtered sequence reads from cDNA sequencing of Treg cells with mean base quality of 9.2 and mean length of 876.4 bp. Preprocessing and filtering of the data set followed by alignment to the mouse genome (mmlO) resulted in 4,724,442 mappable reads (92.7% with 15% error allowance) corresponding to 29,716 transcripts and 15,645 annotated genes. Functional analysis of 212 transcript expressed at TPM > 0.25 resulted in the enrichment of ''Nonsense Mediated Decay (NMD)", "apoptotic signaling pathway", "Antigen processing" and other significant (<1% FDR) pathways. While splicing analysis revealed hundreds of genes exhibiting multiple splicing isoforms.