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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

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Processing Human Thymic Tissue for Single Cell RNA-Seq.

Justin Le¹, Vi Luan Ha^1,2, Annie Luong¹

¹Cancer and Blood Disease Institute, Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.

|October 28, 2020

Summary

This summary is machine-generated.

This protocol isolates rare CD34+ progenitor cells from human thymus for studying thymopoiesis. It uses advanced cell separation techniques to ensure high purity and viability for single-cell RNA sequencing analysis.

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Area of Science:

Immunology
Developmental Biology
Genomics

Background:

Single cell RNA sequencing (scRNA-seq) requires pure and viable cell populations.
Studying human thymopoiesis is crucial for understanding immune system development.
Thymic progenitor cells, including CD34+ cells, are rare and challenging to isolate.

Purpose of the Study:

To describe a protocol for isolating CD34+ progenitor and CD34- differentiated cell fractions from human post-natal thymic tissue.
To enable downstream applications like single cell RNA sequencing for thymopoiesis research.

Main Methods:

Isolation of progenitor (CD34+) and differentiated (CD34-) cell fractions from human thymic tissue.
Utilizes magnetic-activated cell sorting (MACS) followed by fluorescence-activated cell sorting (FACS) for high-purity isolation.
Protocol optimized for rare cell populations (<1% of total thymic cells).

Main Results:

Successfully isolated highly enriched populations of CD34+ progenitor cells.
Obtained distinct CD34- fractions representing more differentiated thymocytes.
Protocol ensures cell viability crucial for downstream scRNA-seq.

Conclusions:

This protocol provides a reliable method for isolating key cell populations from human thymus.
Enables detailed investigation of thymopoiesis at the single-cell level.
Facilitates research into immune cell development and potential therapeutic targets.