Large-scale low-cost NGS library preparation using a robust Tn5 purification and tagmentation protocol [i5i7]

In recent years, tagmentation-based library preparation using a hyperactive version of the Tn5 transposase gained more and more popularity. The limited hands-on time, robustness and high efficiency of the method are essential for the processing of next-generation sequencing libraries from little input material like single cells or the processing of hundreds of samples simultaneously. The hyperactive Tn5 is commercially available (Nextera XT DNA library preparation kit), however, high-throughput experiments with hundreds of samples are costly. Here, we present a highly reproducible Tn5 transposase purification strategy via an N-terminal His6-Sumo3 tag and the workflow for the tagmentation-based NGS library preparation. We demonstrate that NGS libraries processed with the in-house produced Tn5 are of the same quality like those processed with the Nextera XT DNA library preparation kit and that the purification of the transposase is reproducible across institutes. Producing the Tn5 transposase in-house allows for customized experimental design and reduces costs of large-scale experiments dramatically. We describe a novel single cell polyadenylation site mapping protocol that benefits from the fact that the in-house produced Tn5 can be loaded with any desired linker oligonucleotide for tagmentation. Overall design: For samples labeled i5i7, full-length cDNA was amplified from 15pg of HeLa RNA using the smart-seq2 protocol. 75-225pg of cDNA was tagmented using various conditions as outlined in the sample specification. Samples were sequenced using a dual-indexed single read strategy, unless specified differently. Technical tagmentation replicates were processed from the same cDNA input.