High-Throughput Cellular RNA Sequencing (HiCAR-Seq): Cost-Effective, High-Throughput 3' mRNA-Seq Method Enabling...

Area of Science:

• Genomics
• Molecular Biology
• Drug Discovery

Background:

• High-throughput screening is crucial for drug development.
• Transcriptome profiling aids in understanding drug mechanisms, off-target effects, and cytotoxicity.
• Existing high-throughput RNA-Seq (HT RNA-Seq) methods lack sample-wise library quality control.

Purpose of the Study:

• To introduce High-throughput CellulAr RNA Sequencing (HiCAR-Seq), a novel method for transcriptome profiling.
• To enable sample-wise quality control in high-throughput RNA sequencing.
• To provide a robust and efficient method for drug screening and mechanism of action studies.

Main Methods:

• HiCAR-Seq works directly on cultured cells (≥1,000 cells) or 10 ng total RNA.
• It involves oligo-dT reverse transcription, PCR amplification with sample-specific barcodes, and Bioanalyzer verification.
• Libraries are prepared for Illumina sequencing using a tagmentation kit, targeting the 3' end of mRNAs.

Main Results:

• HiCAR-Seq allows for precise cDNA amplification verification and accurate sample pooling.
• As few as 3-4 million reads/sample are sufficient for gene expression analysis in human or mouse cells.
• The method is optimized for direct use on cultured cells or total RNA.

Conclusions:

• HiCAR-Seq offers a robust and competitive alternative to existing HT RNA-Seq methods.
• The ability to perform sample-wise quality control improves data reliability.
• HiCAR-Seq facilitates deeper insights into drug effects through efficient transcriptome profiling.