Key Techniques

What is flow cytometry?

A technique that measures physical and chemical characteristics of individual cells as they pass through a laser beam in a fluid stream. It identifies, counts, and sorts cells based on size, granularity, and fluorescent-labelled markers.

What is flow cytometry used for?

Protein expression

When to use FC?

To identify, quantify, or sort cells by protein markers

Steps of FC

Sample prep, Hydrodynamic focusing, Laser excitation, Optical detection (FSC, SSC, fluorescence), Data acquisition and gating, Compensation, Analysis.

Pros of FC

Rapid, high throughput, multiparametric, quantitative and can physically sort cells,real-time visualisation, and single-cell resolution 

Cons of FC

Requires single-cell suspension, limited number of fluorochromes due to spectral overlap, needs careful compensation and ontrols, expensive and complex

What is MS?

Analytical technique that identifies and quantifies molecules by measuring their m/z ratio

What is MS used for?

Protein/metabolites mass and abundance

When to use MS?

To identify and quantify molecules in a complex sample

Main Steps of MS:

Sample, Ionisation, Ion separation by m/z, Detection, Data analysis (identification, quantification).

Pros of MS:

Highly sensitive and specific, can identify unknown compounds and post-translational modifications, quantitative and reproducible, broad dynamic range

Cons of MS

Complex sample prep and data analysis, matrix effects/ion suppression can bias quantification, dynamic range can miss low-abundance proteins, expensive

What is Single-cell sequencing?

A sequencing technique that profiles gene expression in individual cells to reveal cellular heterogeneity and subpopulations

When to use scRNA-seq?

To explore heterogeneity and discover new cell types

Main step of scRNA-seq

Sample preparation, cell lysis, mRNA capture, cDNA synthesis ad amplification, library construction and QC, sequencing

Pros of scRNA Seq

Single-cell resolution (cell-to-cell variability is good), identifies rare or novel cell types, enables reconstruction of cell differential trajectories, can integrate with other modalities (like ATAQ-seq)

Cons of scRNA-seq

Expensive and data-intensive, sensitive to dropouts, lower sensitivity compared to bulk, requires complex bioinformatic analysis

What is bulk-RNA-seq?

Sequencing method that measures the average gene expression across all cells in a sample

When to use bulkRNA-seq?

To compare overall gene expression between conditions

Main steps of bulkRNA-seq

Experiment setup, sample and library preparation, sequencing, data management, downstream analysis validation

Pros of Bulk-RNA-seq

High reproducibility, deep coverage and good sensitivity, lower cost than scRNA-seq, good for identifying broad transcriptional changes

Cons of bulk-RNA-seq

Averages signals (masks heterogeneity), contaminating cells can skew results, cannot detect rare cell types or subpopulations,  limited to population-level inference

What is NGS

High-throughput sequencing technology that allows millions of DNA or RNA fragments to be sequenced simultaneously

Steps of NGS

Sample collection, dsDNA synthesis, Adapter ligation, Amplification, Sequencing, Read mapping