Achieving high throughput sequencing of a cDNA library utilizing an alternative protocol for the bench top next-generation sequencing system

The development of next-generation sequencing (NGS) technologies has provided novel tools for genome analysis and expression profiling. A high throughput cDNA sequencing method using a bench top next-generation sequencing system, GS Junior, is now available. Here, we used an alternative protocol to the standard method for generating the cDNA library. This protocol can decrease the number of processing steps to manipulate RNA when constructing a cDNA library from an RNA sample, and does not require mRNA isolation from total RNA. Thus it can decrease the risk of RNA degradation and the cost for preparing a cDNA library. Also, the efficiency of sequencing data obtained with this approach is comparable to the standard method as verified by sequencing characteristics and expression levels of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

► We report alternative methods of cDNA library preparation for sequencing utilizing the GS Junior system.
► This protocol can decrease the number of processing steps required to sequence mRNA and prevent degradation.
► This protocol without the need to remove ribosomal RNA can yield high throughput RNA sequencing analysis.
► We represent an effective method for measuring the level of gene expression.