title: Tn5 transposase and tagmentation procedures for massively scaled sequencing projects.
abstract: Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large-scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from subpicogram amounts of cDNA. The comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, because naked Tn5 can be annealed to any oligonucleotide of choice, for example, molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enable innovation in sequencing-based applications.
authors: Ludwig Institute for Cancer Research, 171 77 Stockholm, Sweden;
authors: Simone
authors: S
authors: Picelli
authors: Ludwig Institute for Cancer Research, 171 77 Stockholm, Sweden; Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden.
authors: Asa K
authors: AK
authors: Björklund
authors: Ludwig Institute for Cancer Research, 171 77 Stockholm, Sweden; Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden.
authors: Björn
authors: B
authors: Reinius
authors: Ludwig Institute for Cancer Research, 171 77 Stockholm, Sweden; Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden.
authors: Sven
authors: S
authors: Sagasser
authors: Ludwig Institute for Cancer Research, 171 77 Stockholm, Sweden; Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden.
authors: Gösta
authors: G
authors: Winberg
authors: Ludwig Institute for Cancer Research, 171 77 Stockholm, Sweden; Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden Rickard.Sandberg@ki.se.
authors: Rickard
authors: R
authors: Sandberg
mesh_terms: D018076:DNA, Complementary; D015870:Gene Expression; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA; D059010:Single-Cell Analysis; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; C473503:Tn5 transposase; D019895:Transposases