This month the Research Resource Nexus continues our series on the Center for Advanced Genomics, an umbrella website designed to help investigators determine which of the member cores is the best fit for 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
- 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.
Single Cell Core
The Single Cell Core supports all 10X Genomics single-cell applications on Chromium and Chromium iX controllers.
Available Applications
- Universal Gene Expression
- 3’ or 5’ whole-transcriptome analysis, including isoforms and single nucleotide polymorphisms (SNPs)
- FLEX Gene Expression
- A probe-based method for protein-coding transcriptomics on fixed cells
- compatible with human or mouse cells.
- Epigenomic Assay for Transposase-Accessible Chromatin (Epi-ATAC)
- Examines the open chromatin in single nuclei
- Epi Multiome kit combines 3' RNA expression with ATAC data
- CRISPR Screening
- Identifies the guide RNA (sgRNA) in each cell
- Merges sgRNA information with gene expression profiles in each cell to assess perturbation effects
- Multiomic Add-Ons
- Cell surface proteins, to assess cell subtypes and states, can be combined with any universal or FLEX gene expression analysis.
- Intracellular protein levels can be measured in parallel with gene expression using the FLEX kit.
- CRISPR status of each cell can be combined with 5’ or FLEX gene expression data.
- Full-length T and/or B cell receptor profiles can be enriched from 5’ gene expression cDNA. Combined analysis provides a detailed picture of the immune system.
On-chip multiplexing is now available for all Universal Gene Expression kits. It is a cost-effective approach for pilot projects and samples with limited cell numbers. This new application works on the Chromium or Chromium iX chip to combine the gel beads in emulsion (GEMs) for up to four samples.
Quality sample preparation is a crucial component of single-cell analysis and is the researcher’s responsibility. Cell suspensions with high percent viability must be in appropriate buffers with minimal clumps and debris. Detailed guidance is available under the “Preparing Samples for Submission” section of the Advanced Genomics Resources page. Consult the experienced staff at the Single Cell Core to optimize your cell preparation before scheduling your sequencing submission.
Prompt sample processing is essential to maintaining cell viability for robust, reproducible and meaningful data. Live cell drop-offs must be coordinated with core lab staff to ensure initial steps can proceed immediately. Fixed cells may be submitted at any time.
Bioinformatic support is available through the Single Cell Core and other resources, like the Genomics Analysis Core.
Contact the Single Cell Core to initiate your next project.
Selected Projects
- Vascularization—the process by which blood vessels form and grow within a tissue—is essential for organ maturation and developing organ-specific cell types. Successful drug discovery, organ modeling and eventual organ transplantation depend on achieving robust vascular networks within lab-grown organs. Working within a kidney organoid model, Neil Hukriede’s lab used the Single Cell core to characterize a transgenic cell line and to investigate the cellular composition of resulting vascularized organoids in their work integrating a human induced pluripotent stem cell (iPSC) line with a nontransgenic iPSC line in suspension organoid cultures. This study advances the field of kidney tissue engineering by bringing the field significantly closer to more accurate and functional disease modeling.1
- Harinder Singh’s lab used scRNA-seq with cellularly paired B Cell Receptor sequencing (BCR-seq) to examine the developmental trajectory whereby B cells in germinal centers transition to long-lived bone marrow plasma cells (PCs). They identified an intermediate cell state in the transition from B cells to PCs called PC progenitor. After clonal expansion (when an immune cell makes many copies of itself), each resulting PC progenitor was found to further differentiate into one of three types of plasma cells. PCs characterized by the expression of Tigit predominate in the bone marrow, giving rise to long-lived bone marrow PCs. The use of single-cell multiomics, combining RNA-seq with BCR-seq for clonal identification, was essential to this work.2
1. Maggiore JC, LeGraw R, Przepiorski A, Velazquez J, Chaney C, Vanichapol T, Streeter E, Almuallim Z, Oda A, Chiba T, Silva-Barbosa A, Franks J, Hislop J, Hill A, Wu H, Pfister K, Howden SE, Watkins SC, Little MH, Humphreys BD, Kiani S, Watson A, Stolz DB, Davidson AJ, Carroll T, Cleaver O, Sims-Lucas S, Ebrahimkhani MR, Hukriede NA. A genetically inducible endothelial niche enables vascularization of human kidney organoids with multilineage maturation and emergence of renin expressing cells. Kidney Int. 2024 Dec;106(6):1086-1100. doi: 10.1016/j.kint.2024.05.026. Epub 2024 Jun 18. PMID: 38901605; PMCID: PMC11912416.
2. Manakkat Vijay GK, Zhou M, Thakkar K, Rothrauff A, Chawla AS, Chen D, Lau LC, Gerges PH, Chetal K, Chhibbar P, Fan J, Das J, Joglekar A, Borghesi L, Salomonis N, Xu H, Singh H. Temporal dynamics and genomic programming of plasma cell fates. Nat Immunol. 2024 Jun;25(6):1097-1109. doi: 10.1038/s41590-024-01831-y. Epub 2024 May 2. PMID: 38698087.