| Autor: |
Schönegger ES; Ludwig-Maximilians-Universität München, Institute for Chemical Epigenetics Munich, Butenandtstr. 5-13, 81377 Munich, Germany., Crisp A; baseclick GmbH, Floriansbogen 2-4, 82061 Neuried (Munich), Germany., Müller M; Ludwig-Maximilians-Universität München, Institute for Chemical Epigenetics Munich, Butenandtstr. 5-13, 81377 Munich, Germany., Fertl J; baseclick GmbH, Floriansbogen 2-4, 82061 Neuried (Munich), Germany., Serdjukow S; baseclick GmbH, Floriansbogen 2-4, 82061 Neuried (Munich), Germany., Croce S; baseclick GmbH, Floriansbogen 2-4, 82061 Neuried (Munich), Germany., Kollaschinski M; baseclick GmbH, Floriansbogen 2-4, 82061 Neuried (Munich), Germany., Carell T; Ludwig-Maximilians-Universität München, Institute for Chemical Epigenetics Munich, Butenandtstr. 5-13, 81377 Munich, Germany., Frischmuth T; baseclick GmbH, Floriansbogen 2-4, 82061 Neuried (Munich), Germany. |
| Abstrakt: |
Here we describe the development of a novel click chemistry-based method for the generation and amplification of full-length cDNA libraries from total RNA, while avoiding the need for problematic template-switching (TS) reactions. Compared with prior efforts, our method involves neither random priming nor stochastic cDNA termination, thus enabling amplification of transcripts that were previously inaccessible via related click chemistry-based RNA sequencing techniques. A key modification involving the use of PCR primers containing two overhanging 3'-nucleotides substantially improved the read-through compatibility of the 1,4-disubstituted 1,2,3-triazole-containing cDNA, where such modifications typically hinder amplification. This allowed us to more than double the possible insert size compared with the state-of-the art click chemistry-based technique, PAC-seq. Furthermore, our method performed on par with a commercially available PCR-cDNA RNA sequencing kit, as determined by Oxford Nanopore sequencing. Given the known advantages of PAC-seq, namely, suppression of PCR artifacts, we anticipate that our contribution could enable diverse applications including improved analyses of mRNA splicing variants and fusion transcripts. |
