RNA-Seq Library Generation from Rare Human Cells Isolated by FACS

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• High-throughput RNA Sequencing (RNA-Seq) is crucial for transcriptome analysis.
• Existing protocols require microgram quantities of RNA, limiting studies on rare or scarce cell populations.
• Analyzing small cell numbers is challenging due to RNA quantity requirements.

Purpose of the Study:

• To develop a method for preparing RNA sense-strand cDNA libraries for Illumina sequencing from limited numbers of human cells.
• To enable transcriptome analysis of rare cell types, such as human fetal germ cells and human embryonic stem cells (hESCs).

Main Methods:

• Utilized an RNA Amplification System.
• Prepared RNA sense-strand cDNA libraries compatible with the Illumina sequencing platform.
• Isolated cells using Fluorescence Activated Cell Sorting (FACS).

Main Results:

• Successfully generated seven RNA-Seq libraries from 4,000 sorted human fetal germ cells (from ovaries and testes) and 4,000 sorted hESCs.
• Demonstrated the feasibility of the protocol for low-input RNA samples.

Conclusions:

• The described protocol enables RNA sequencing from limited cell numbers, expanding the scope of transcriptome analysis.
• The method is applicable to precious rare cell types and can be adapted for multiplexed library generation.