Abstract
Despite its widespread use, RNA-seq is still too laborious and expensive to replace RT-qPCR as the default gene expression analysis method. We present a novel approach, BRB-seq, which uses early multiplexing to produce 3′ cDNA libraries for dozens of samples, requiring just 2 hours of hands-on time. BRB-seq has a comparable performance to the standard TruSeq approach while showing greater tolerance for lower RNA quality and being up to 25 times cheaper. We anticipate that BRB-seq will transform basic laboratory practice given its capacity to generate genome-wide transcriptomic data at a similar cost as profiling four genes using RT-qPCR.
Highlights
High-throughput sequencing has become the method of choice for genome-wide transcriptomic analyses as its price has substantially decreased over the last years
Adaptation of the early multiplexing RNA-seq library preparation workflow First, we set out to benchmark SCRB-seq against the “gold standard” Illumina TruSeq workflow for bulk gene expression profiling
We prepared libraries following both protocols using RNA from GM12878 cells treated with either DMSO or IKK inhibitor (BAY 11-7082) to induce gene expression differences and to assess a potential difference between these two methods in the power to detect differentially expressed genes starting from the same RNA
Summary
High-throughput sequencing has become the method of choice for genome-wide transcriptomic analyses as its price has substantially decreased over the last years. The high cost of standard RNA library preparation and the complexity of the underlying data analysis still prevent this approach from becoming as routine as quantitative (q) PCR, especially when many samples need to be analyzed. To alleviate this high cost, the emerging single-cell transcriptomics field implemented the sample barcoding/early multiplexing principle. This reduces both the RNA-seq cost and preparation time by allowing the generation of a single sequencing library that contains multiple distinct samples/ cells [1]. There have been surprisingly few efforts to explicitly adapt and validate the early-stage multiplexing protocols for reliable and cheap profiling of bulk RNA samples
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