Core B: Single Cell Protein and RNA Sequencing Core

核心B概述

Core B Summary Core B serves all 4 projects in this PPG for all RNA sequencing needs, including bulk and single cell RNA- sequencing (scRNA-Seq), protein sequencing by oligonucleotide-tagged antibodies (Ab-Seq), T- and B-cell receptor sequencing (TCR-Seq and BCR-Seq) and ATAC-Seq. Core B is responsible for all quality controls along the workflow. Core B staff consists of wet lab staff and bioinformaticians. They interact with postdocs, technicians, epidemiologists and PIs from the Human Clinical Cardiovascular and Biostatistics Core (Clinical Core C) and from each of the four projects. Workflows are established for mouse (macrophages, sorted T cells) and human (PBMC) cells. Hashtags and antibodies are fully validated, including thresholding, which improves data quality by eliminating signal from unbound antibody and non-specific antibody binding. The previous version of this core (core E in the 2016 Hedrick PPG) was the first group world-wide to use scRNA- Seq in atherosclerosis research. We established and published guidelines for workflows and quality controls in scRNA-Seq, batch effect, doublet and dead cell removal. Core B provides wet lab and bioinformatics service plus well-developed interfaces with all 4 projects and core C. For Ab-Seq, we validated a 192 oligonucleotide- tagged human antibody panel (Biolegend), run on 10x Genomics 5’, which will be used for Ab-Seq in most of the proposed human studies. For mice, we use the Biolegend 138 mouse antibody panel with 10x Genomics 5’. Using this platform, we have successfully assembled tens of thousands of TCRα and β chain pairs (scTCR- Seq) and BCR heavy and light chain pairs (scBCR-Seq). All 4 projects use core B. The specific aims are: (1) To provide the highest quality scRNA-Seq, scAb-Seq, scATAC-Seq, scTCR-Seq and scBCR-Seq for mouse and human samples, including fully documented and rigorous quality controls, designing and optimizing antibody panels and advice on all bioinformatics issues. (2) To provide the highest quality bulk RNA-Seq and ATAC-Seq for mouse and human samples, including fully documented and rigorous quality controls and support for experimental design and planning. (3) To effectively and seamlessly interface with postdocs, technicians, epidemiologists and PIs from the Clinical Core C and from each of the four projects, including sample shipping, receiving, thawing, data processing, visualization and delivery. To provide these services, we have built a team of wet lab technicians, bioinformaticians and computational biologists. Interfaces with all projects and core C are established and working well.