# Core E: Cell sorting, CyTOF and RNA-Seq

> **NIH NIH P01** · LA JOLLA INSTITUTE FOR IMMUNOLOGY · 2021 · $544,461

## Abstract

Core E description
Core E has four functions, serving all 4 projects. First, human and mouse T cells, B cells and monocytes will be
sorted from peripheral blood mononuclear cells (PBMCs) and sorted into trizol, RNA extracted, quality controls
(bioanalyzer, Agilent TapeStation) performed, libraries prepared and RNA-Seq done to obtain ~50-70 million
Illumina reads per sample. Preliminary data show excellent sequencing depth and data quality even in long-
term frozen PBMCs. Mouse and human samples are focused on monocyte subsets for project 1, treated
human and sorted mouse macrophages for project 2, B1 cells from peritoneal cavity, spleen and bone marrow
in wild-type, Cxcr4-/- and Cxcr5-/- mice for project 3, and dextramerhigh, dextramerlow, dextramernegative effector
memory (TEM) cells and naïve CD4 T cells for project 4. Second, 36 to 42-parameter mass cytometry (CyTOF)
followed by high-dimensional analysis (SPADE, viSNE) will be used for unsupervised clustering. We have
already acquired or conjugated, titrated and validated more than 100 monoclonal antibodies, developed one
human panel that will serve all projects and four human panels that serve each project individually. We will use
about 1 million cells from each tube for this, and the remaining about 7 million cells will be used for RNA-Seq.
We also will run CyTOF on mouse. CyTOF identifies the known cell types and likely will suggest new, unknown
cell types (new subsets of monocytes, B cells, T cells). Third, sorted T cells and antigen presenting cells
(APCs) will be distributed to project 4 and sorted monocytes to project 1 for functional experiments in vivo and
in vitro as detailed in the projects. Finally, core E will also provide basic bioinformatics support. This includes
post-sequencing quality controls for RNA-Seq, mapping reads, differential expression (DE), heat maps, Venn
diagrams and principal component analysis (PCA).

## Key facts

- **NIH application ID:** 10188604
- **Project number:** 5P01HL136275-05
- **Recipient organization:** LA JOLLA INSTITUTE FOR IMMUNOLOGY
- **Principal Investigator:** Klaus F. Ley
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $544,461
- **Award type:** 5
- **Project period:** 2017-08-01 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10188604

## Citation

> US National Institutes of Health, RePORTER application 10188604, Core E: Cell sorting, CyTOF and RNA-Seq (5P01HL136275-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10188604. Licensed CC0.

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