HCA Data Explorer

A Protocol for Revealing Oral Neutrophil Heterogeneity by Single-Cell Immune Profiling in Human Saliva

Updated August 30, 2022

Neutrophils are the most abundant white blood cells in the human body responsible for fighting viral, bacterial and fungi infections. Out of the 100 billion neutrophils produced daily, it is estimated that 10 % of these cells end up in oral biofluids. Because saliva is a fluid accessible through non-invasive techniques, it is an optimal source of cells and molecule surveillance in health and disease. While neutrophils are abundant in saliva, scientific advancements in neutrophil biology have been hampered likely due to their short life span, inability to divide once terminally differentiated, sensitivity to physical stress, and low RNA content. Here, we devise a protocol aiming to understand neutrophil heterogeneity by improving isolation methods, single-cell RNA extraction, sequencing and bioinformatic pipelines. Advanced flow cytometry 3D analysis, and machine learning validated our gating system model, by including positive neutrophil markers and excluding other immune cells and uncovered neutrophil heterogeneity. Considering specific cell markers, unique mitochondrial content, stringent and less stringent filtering strategies, our transcriptome single cell findings unraveled novel neutrophil subpopulations. Collectively, this methodology accelerates the discovery of salivary immune landscapes, with the promise of improving the understanding of diversification mechanisms, clinical diagnostics in health and disease, and guide targeted therapies.

Marcelo FreireJ. Craig Venter Institute
Saibyasachi Choudhury1
Mark Novotny1
Brian Aevermann1
Steven Lee1
Aishwarya Mandava1
Yu Qian1
Richard Scheuermann1
Marcelo Freire (Principal Investigator)1
1J. Craig Venter Institute
Ray Stefancsik

To reference this project, please use the following link:

https://explore.data.humancellatlas.dev.clevercanary.com/projects/60ea42e1-af49-42f5-8164-d641fdb696bc
None
INSDC Project Accessions:INSDC Study Accessions:

Atlas

None

Analysis Portals

None

Project Label

PRJNA640427_human_neutrophils

Species

Homo sapiens

Sample Type

specimens

Anatomical Entity

oral cavity

Organ Part

saliva

Selected Cell Types

neutrophil

Disease Status (Specimen)

normal

Disease Status (Donor)

normal

Development Stage

human adult stage

Library Construction Method

Smart-seq2

Nucleic Acid Source

single cell

Paired End

true

File Format

2 file formats

Cell Count Estimate

1.1k

Donor Count

3
fastq.gz2,290 file(s)xlsx1 file(s)