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Old 07-31-2014, 07:49 AM   #1
Simone78
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Default "home-made" Tn5 transposase for single-cell RNA-seq

here is our paper on Tn5 transposase production which doesn´t require the purchase of an expensive Nextera kit from Illumina:

http://genome.cshlp.org/content/earl....full.pdf+html

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 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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Old 08-05-2014, 10:37 PM   #2
SNPsaurus
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Nice work! Lots of great details beyond the DIY aspect.
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Old 08-21-2014, 01:38 PM   #3
Luka
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Nice work Simone!

I would just have one question why did you decide to have your TN5 wild type with respect to the M56 mutation?

Thanks
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nextera sample prep, rna-seq, single cell, tn5

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