ClickSeq is a simple, fragmentation-free method for the synthesis of Next-Generation Sequencing (NGS) libraries. ClickSeq derives its name by using ‘Click-Chemistry‘ in the place of common ligation enzymes to ‘click-ligate’ nucleic acids to sequencing adaptors – an essential and often problematic step in the synthesis of Next-Generation Sequencing cDNA libraries. The process takes advantage of the chain-terminating properties of 3′-azido-nucleotides, which are included the initial in vitro reverse-transcription reactions uniformly required for RNAseq. The modified nucleotides are stochastically incorporated into the nascent cDNA, yielding cDNA fragments blocked at their 3′ ends with azido groups. The 3′-azido-blocked cDNA fragments are ‘click-ligated’ onto alkyne-functionalized sequencing adaptors, which can subsequently be PCR-amplified to yield a sequencing-ready NGS library. ClickSeq is a highly flexible and modular platform for NGS library synthesis where both RNA and DNA templates can be used as input material. ClickSeq offers many advantages over standard RNA NGS protocols as click-chemistry is utilized to attach on the required sequencing adapter, rather than commonly used enzymatic reactions. Overall, this results in increased efficiency of the protocol, fewer processing steps, and reduced time from RNA to sequencing ready libraries. Additionally, since there are no fragmentation steps typically required in common RNA-seq approaches and because in vitro template switching during RT is limited by the chain terminating azido-nucleotides, ClickSeq offers the benefit of ultra-low artifactual chimera rate, with only 3 chimeric events per million reads.