Tn5 transposase and tagmentation procedures for massively scaled sequencing projects
By S. Picelli, Åsa K. Björklund, Björn Reinius, Sven Sagasser, Gösta Winberg, Rickard Sandberg. Genome Research, 2014
810 citations recorded by the source corpus.
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.
Subfields: Molecular Biology
View Paper: https://www.semanticscholar.org/paper/99c82bdd33a574954d6b7825585cde61a8b3c53e
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In Molecular Biology
- Aviv Regev
- Caleb A. Lareau
- Jiarui Ding
- Rickard Sandberg
- S. Picelli
- Eran Elinav
- Bin Tian
- Hadas Keren-Shaul
Citations in the corpus
- Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin (2020, methodology): “Transposome preparation— Tn5 was produced in-house by following a published protocol with minor modifications (Picelli et al., 2014).”
- Single-Cell Transcriptome Profiling of Human Pancreatic Islets in Health and Type 2 Diabetes (2016, methodology): “Tn5 transposase directed tagmentation of 0.5-1 ng cDNA was performed with recombinant Tn5 in 10% PEG8000, 10mM TAPS-NaOH (pH 8.3), 5mM MgCl2 (Picelli et al., 2014b).”
- RNA-Seq methods for transcriptome analysis (2016, methodology): “Recent development in using transposon-based, so-called tagmentation method has made it simple to fragment cDNA and add adapter sequences at the same time.(25) In this method, an active variant of the Tn5 transposase mediates the fragmentation of double-stranded DNA and ligates adapter oligonucleoti”
- Microglia development follows a stepwise program to regulate brain homeostasis (2016, methodology): “To profile for open chromatin, we used an adaptation of the ATAC-seq protocol (46, 82) as previously described (16).”