ClickSeq follows the same principle of all NGS library preps that seek to generate short fragments of cDNAs that are flanked with the appropriate sequencing adaptor for the user’s choice of sequencing platform (e.g. Illumina).

The process works by supplementing the Reverse Transcription step of an NGS library prep with small amounts of terminating ‘azido-nucleotides’. These are stochastically incorporated into the cDNA during first stranded synthesis, yielding 3’azido-terminated cDNA molecules. The azido group is then ‘Click-Ligated’ to an alkyne-functionalized sequencing adaptor using Click-Chemistry (Copper Catalysed Azide-Alkyne Cycloaddition, CuAAC). The resultant click-linked single-stranded cDNA is then PCR amplified to generate a final sequencing-ready NGS library.

Yes. Whatever you put into your NGS prep is what you will sequence, with the exception of very short RNAs. Since ClickSeq relies on the stochastic incorporation of azido-nucleotides into cDNA during reverse transcription, the reverse transcriptase might reach the end of small RNAs before an azido-nucleotide is incorporated. As a result, the cDNA cannot be click-ligated to the sequencing adaptor and thus will not be sequenced.

Yes. ClickSeq can sequence DNA with the exact same protocol as used for RNAseq provided that the RT enzyme has DNA-templated DNA polymerase activity (such as SuperScript III). Published examples of this can be found here:

https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giad009/7080818

Yes. ClickSeq was originally published in 2015 by Routh et al in the Journal of Molecular Biology:

https://www.sciencedirect.com/science/article/abs/pii/S0022283615003514?via%3Dihub

ClickSeq has since been used in numerous publications, by the original inventors, by clients who have purchased ClickSeq kits and/or services as well as independent groups who have assembled their own reagents. See here for a curated list of publications:

https://www.clickseqtechnologies.com/resources

Our protocol can be found here:

https://www.protocols.io/view/clickseq-random-primed-protocol-with-single-indexi-n92ld8jkov5b/v1

Required third-party reagents include:

• SuperScript III™ Reverse Transcriptase, 200U/µL (Invitrogen; 18080-093 or 18080-044)
• [optional] RNaseOUT™ Recombinant Ribonuclease Inhibitor, 40U/µL (Invitrogen; 10777)
• OneTaq® 2X Master Mix with Standard Buffer (NEB; M0482S or M0482L) (Note: you must use OneTaq for this step, this enzyme cannot be substituted for a different PCR enzyme)
• [optional] RNase H, 5000 units/mL (NEB; M0297S or M0297L)
• SPRIselect (Beckman Coulter; B23317) or equivalent DNA/RNA Purification Beads (also known as SPRI beads)
• Nuclease free water
• 80% ethanol (made fresh)

Please see an extensive set of FAQs:

https://www.clickseqtechnologies.com/resources/faqs