3' mRNA-seq is a quantitative, genome-wide transcriptomic technique based on the barcoding of the 3' untranslated region (UTR) of mRNA molecules. Unlike standard bulk RNA-seq, where short sequencing reads are generated along the entire length of mRNA transcripts, only the 3' end of polyadenylated RNAs are sequenced in 3' mRNA-seq. This approach results in a need for fewer reads to quantify the expression of a gene and reduces the sequencing depth required per sample while providing robust and reliable transcriptome-wide read-outs of gene expression levels comparable to full-length RNA-seq methods.[1][2]
Sample barcoding and the reduced per-sample sequencing depth also allow higher levels of sample multiplexing per experiment and lower the cost of transcriptome sequencing compared to full-length RNA-seq methods. These factors are crucial for large-scale, ultra-high-throughput gene expression studies or studies assessing differential gene expression between different experimental conditions or cell types.[2]
Some 3' mRNA-seq technologies, like Bulk RNA Barcoding and Sequencing (BRB-seq) commercialized by Alithea Genomics further streamline the library preparation process by pooling up to 384 samples very early in the workflow for a cost per sample tantamount to profiling four individual genes using conventional qRT-PCR, in a workflow requiring less than two and a half hours hands-on time.[2] An increasing number of 3' mRNA-seq techniques also include unique molecular identifiers (UMIs) in sample barcodes to uniquely label each mRNA molecule and to distinguish between original mRNA transcripts and duplicates that result from PCR amplification.