Last month the Research Resource Nexus introduced the Center for Advanced Genomics—a centralized website designed to help investigators determine which of the center’s member cores best fit their project needs. Member cores include
- the Health Sciences Sequencing Core at UPMC Children’s Hospital of Pittsburgh
- for low-input RNA-seq, spatial biology, ChIP-seq or ATAC-seq and high-throughput sequencing;
- the High-Throughput Genomics Core
- for whole genome or whole exome sequencing, high-input high-throughput RNA-seq, Axiom arrays, clinical testing and high-throughput sequencing; and
- the Single Cell Core
- for multiple types of single cell analyses.
To initiate a project through the Center for Advanced Genomics, or if you have general inquiries, fill out a project inquiry form. Staff from the appropriate genomic core facility will respond.
This month’s edition spotlights the Health Sciences Sequencing Core at UPMC Children’s Hospital of Pittsburgh (HSSC). HSSC operates high-throughput Illumina sequencers and offers expert support in RNA/DNA extraction and multiple library preparation services. Experienced staff and validated workflows support a diverse portfolio of applications.
Health Sciences Sequencing Core (HSSC)
HSSC offers a full complement of services supporting bulk RNA-seq including
- standard- and ultralow-input from RNA (poly A+) or total RNA,
- small/microRNA, and
- T cell receptor (TCR) profiling.
Spatial transcriptomics are available on Curio Slide Seeker and 10X Genomics platforms, including Visium, VisiumHD and Xenium. These technologies support spatial transcriptomic analysis of
- fresh frozen tissue from any species (Visium and Curio Slide Seeker platforms), and
- fresh/fixed frozen or Formalin-Fixed Paraffin-Embedded (FFPE) human or murine tissue (Visium, VisiumHD and Xenium platforms).
With 10x Genomics CytAssist, HSSC can transfer sections from standard slides to specialized, barcoded Visium and VisiumHD slides that provide spatial resolution. In addition, HSSC can perform mouse or human TCR or B cell receptor spatial sequencing using their fresh frozen platforms.
Epigenetics is the study of chromatin modifications and interactions. HSSC offers support for selected sequencing-based epigenetic applications.
- ATAC-seq (Assay for Transposase Accessible Chromatin) assesses open chromatin in living cells. Using HSSC for this library preparation method requires close coordination between the submitting laboratory and HSSC staff to ensure processing can proceed while cells are viable and unstressed.
- ChIP-seq (Chromatin ImmunoPrecipitation) allows investigators to identify DNA targets of known transcription factors or other DNA-binding proteins. Immunoprecipitation is done in the primary investigator’s (PI) lab and the product delivered to HSSC for careful and expert library preparation of these precious ultralow-input samples.
- CUT&RUN or CUT&TAG (low-input ChIP-seq-like assay) allows investigators to identify DNA targets of known transcription factors or other DNA binding proteins from very low cell counts (500-100,000 cells). CUT&RUN or CUT&TAG protocols are done in the PI’s lab. Libraries are delivered to HSSC for quantitation, pooling and sequencing.
HSSC also offers DNA/RNA extraction from
- cell pellets, including buffy coat;
- fresh frozen or FFPE tissue;
- saliva; and
- whole blood, serum or plasma.
For skilled laboratories that prefer to do their own benchwork, HSSC accepts prepared libraries compatible with the NextSeq2000 or MiSeq instruments.
To initiate a project with the HSSC, complete the project inquiry form.
Selected Projects and Testimonials
Sarah Hainer (Department of Biological Sciences) shared: “In the Hainer lab, we are interested in understanding fundamental epigenomic mechanisms that drive cell identity. The 200-plus cell types found in the adult organism all have the same DNA sequence but have specified functions. Therefore, it is not the DNA sequence but rather the utility of the DNA sequence that drives identity. For example, in a neuron, neural-specific transmitter proteins are required to be transcribed and translated for appropriate function, but these same genes are silent in liver cells. Our lab uses functional genomic approaches to understand these regulatory mechanisms. We employ technologies including RNA-seq, nascent RNA-seq, scRNA-seq, CUT&RUN, ATAC-seq, MNase-seq and HiC to achieve these goals. We work with HSSC for all our sequencing needs. We have always been impressed with the speed, quality and ease of communication with HSSC. Specifically, Assistant Director Will McDonald is extremely knowledgeable and helpful in setting up new projects and working to achieve our goals.”
Radosveta Koldamova (Department of Environmental and Occupational Health) used multitranscriptomics (bulk, single cell and spatial) to investigate the mechanisms linking peripheral inflammation to Alzheimer’s disease pathology in a mouse model of the disease. Using HSSC services for spatial transcriptomics on the Visium platform Koldamova’s group identified gene expression changes in the plaque microenvironment and at the ventricular surfaces indicating possible disruption of the blood-brain and blood-cerebrospinal fluid barriers due to peripheral inflammation.¹
Jun Chen (Department of Neurology) combined RNA-seq with flow cytometry, imaging and other applications to identify a novel subpopulation of CD8+CD122+ T cells (CD8+CD122+CD49dlo T-regulatory-like cells), which infiltrate the post-ischemic brain in mice, reducing inflammation and providing neuroprotection, thereby limiting infarct expansion. The study relied on HSSC ultralow-input RNA library preparation services to analyze changes in post-ischemic brain infiltrating CD8+ cells and identify mechanisms associated with the beneficial effects of this cell population.² Future research building on this study could lead to new therapeutics and improved outcomes for stroke patients.
1. Lu Y, Saibro-Girardi C, Fitz NF, McGuire MR, Ostach MA, Mamun-Or-Rashid ANM, Lefterov I, Koldamova R. Multi-transcriptomics reveals brain cellular responses to peripheral infection in Alzheimer's disease model mice. Cell Rep. 2023 Jul 25;42(7):112785. doi: 10.1016/j.celrep.2023.112785. Epub 2023 Jul 11. PMID: 37436901; PMCID: PMC10530196.
2. Cai W, Shi L, Zhao J, Xu F, Dufort C, Ye Q, Yang T, Dai X, Lyu J, Jin C, Pu H, Yu F, Hassan S, Sun Z, Zhang W, Hitchens TK, Shi Y, Thomson AW, Leak RK, Hu X, Chen J. Neuroprotection against ischemic stroke requires a specific class of early responder T cells in mice. J Clin Invest. 2022 Aug 1;132(15):e157678. doi: 10.1172/JCI157678. PMID: 35912857; PMCID: PMC9337834.