Project description:Long-read sequencing technologies such as Iso-Seq (PacBio Inc.) generate highly accurate sequences of full-length mRNA transcript isoforms. Long-read transcriptomics may be especially useful in the context of T cell functional plasticity as it relates to human health and disease. However, To our knowledge, no long-read transcriptome reference exists for activated human CD4 T cells. To begin to fill this gap, we purified CD4 T cells from the peripheral blood of a healthy female donor and activated these cells with anti-CD3/CD28 beads to generate populations of early activated (4hr), mid-activated (16hr), blasting (48hr) and proliferating (120hr) CD4 T cells. From each of these time points, we obtained high-quality RNA (RIN>9) for PacBio Iso-Seq analysis and parallel RNA-Seq analysis, which we hope will serve as a reference for future transcriptomic studies of these populations. UCSC genome browser tracks for these samples can be accessed at: http://genome.ucsc.edu/cgi-bin/hgHubConnect?hgHub_do_redirect=on&hgHubConnect.remakeTrackHub=on&hgHub_do_firstDb=on&position=chr1:206,903,317-206,921,941&hubUrl=http://162.215.210.70/~tracks/Mitchell_IsoSeq_Stim/hub.txt
Project description:Long-read sequencing technologies such as Iso-Seq (PacBio Inc.) generate highly accurate sequences of full-length mRNA transcript isoforms. Long-read transcriptomics may be especially useful in the context of T cell functional plasticity as it relates to human health and disease. However, To our knowledge, no long-read transcriptome reference exists for activated human CD4 T cells. To begin to fill this gap, we purified CD4 T cells from the peripheral blood of a healthy female donor and activated these cells with anti-CD3/CD28 beads to generate populations of early activated (4hr), mid-activated (16hr), blasting (48hr) and proliferating (120hr) CD4 T cells. From each of these time points, we obtained high-quality RNA (RIN>9) for PacBio Iso-Seq analysis and parallel RNA-Seq analysis, which we hope will serve as a reference for future transcriptomic studies of these populations. UCSC genome browser tracks for these samples can be accessed at: http://genome.ucsc.edu/cgi-bin/hgHubConnect?hgHub_do_redirect=on&hgHubConnect.remakeTrackHub=on&hgHub_do_firstDb=on&position=chr1:206,903,317-206,921,941&hubUrl=http://162.215.210.70/~tracks/Mitchell_IsoSeq_Stim/hub.txt
Project description:<p>We use next generation sequencing to investigate the different transcriptomes of closely related CD4+ T-cells from healthy human donors to elucidate the genetic programs that underlie their specialized immune functions. Six cell types were included: Regulatory T-cells (CD25hiCD127low/neg with >95% FOXP3+ purity), regulatory T-cells activated using PMA/ionomycin, CD25-CD45RA+ ('naive' helper T-cells), CD25-CD45RO+ ('memory' helper T-cells), activated Th17 cells (>98% IL17A+ purity) and activated IL17-CD4+ T-cells (called 'ThPI'). Poly-T capture beads were used to isolate mRNA from total RNA, and fragment sizes of ~200 were sequenced from both ends on Illumina's genome analyzer. We confirm many of the canonical signature genes of T-cell populations, but also discover new genes whose expression is limited to specific CD4 T-cell lineages, including long non-coding RNAs. Additionally, we find that genes encoded at loci linked to multiple human autoimmune diseases are enriched for preferential expression upon T-cell activation, suggesting that an aberrant response to T-cell activation is fundamental to pathogenesis.</p>
Project description:Long-read sequencing technologies such as Iso-Seq (PacBio Inc.) generate highly accurate sequences of full-length mRNA transcript isoforms. Long-read transcriptomics may be especially useful in the context of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes seem to be publicly available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified four lymphocyte subsets (CD4 T, CD8 T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN>8) for PacBio Iso-Seq analysis and parallel RNA-Seq analysis.
Project description:Long-read sequencing technologies such as Iso-Seq (PacBio Inc.) generate highly accurate sequences of full-length mRNA transcript isoforms. Long-read transcriptomics may be especially useful in the context of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes seem to be publicly available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified four lymphocyte subsets (CD4 T, CD8 T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN>8) for PacBio Iso-Seq analysis and parallel RNA-Seq analysis.