With the rapid development of NGS technologies, RNA-seq has become the new standard for transcriptome analysis. Although the
price per base has been substantially reduced, sample preparation, sequencing and data processing are major cost factors in high throughput screenings.
QuantSeq provides a protocol to generate highly strand-specific next-generation sequencing (NGS) libraries close to the 3’ end of polyadenylated RNAs within 4.5 h. Only one fragment per transcript is generated, directly linking the number of reads mapping to a gene to its expression. QuantSeq reduces data analysis time and enables a higher level of multiplexing per run.
Library generation is initiated by oligo-dT priming (Fig. 1a), and no prior poly(A) enrichment or ribosomal RNA depletion is required. First-strand synthesis and RNA removal is followed by random-primed synthesis of the complementary strand (second-strand synthesis). Illumina- or IonTorrent-specific linker sequences are introduced by the primers. The resulting double-stranded cDNA is purified with magnetic beads, rendering the protocol compatible with automation. Library PCR amplification then introduces the complete sequences required for cluster generation (Fig. 1b). Illumina libraries can be multiplexed with up to 96 external barcodes and are compatible with both single-end and paired-end sequencing reagents. The insert size is optimized for short reads (e.g., SR50 or SR100) while maintaining suitability for longer read lengths. IonTorrent libraries can be multiplexed using 24 in-line barcodes.