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| Status |
Public on Jul 14, 2014 |
| Title |
Tn5 transposase and tagmentation procedures for massively scaled sequencing projects |
| Organisms |
Homo sapiens; Mus musculus; synthetic construct |
| Experiment type |
Expression profiling by high throughput sequencing
|
| Summary |
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 sub-picogram amounts of cDNA. 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, as 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 enables innovation in sequencing-based applications.
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| Overall design |
Sequencing libraries from cDNA was generated using in-house produced or commercial Tn5 and in a variety of tagmentation buffers and conditions.
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| Contributor(s) |
Picelli S, Björklund Å, Sandberg R, Reinius B, Winberg G |
| Citation(s) |
25079858 |
| Submission date |
Jun 19, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Rickard Sandberg |
| E-mail(s) |
Rickard.Sandberg@ki.se
|
| Organization name |
Karolinska Institutet
|
| Department |
Department of Cell and Molecular Biology
|
| Street address |
Berzelius vag 35
|
| City |
Stockholm |
| ZIP/Postal code |
17177 |
| Country |
Sweden |
| |
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| Platforms (3) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
| GPL15228 |
Illumina HiSeq 2000 (synthetic construct) |
|
| Samples (67)
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| Relations |
| BioProject |
PRJNA253099 |
| SRA |
SRP043417 |
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