| Series | GSE58652 |
| Title | Tn5 transposase and tagmentation procedures for massively scaled sequencing projects |
|---|---|
| Year | 2014 |
| Country | Sweden |
| Article | Sandberg R,Winberg G,Sagasser S,Reinius B,Björklund AK,Picelli S.Tn5 transposase and tagmentation procedures for massively scaled sequencing projects.Genome research.2014 Dec |
| PMID | 25079858 |
| Bio Project | BioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA253099 |
| Sra | SRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP043417 |
| Overall Desgin | Sequencing libraries from cDNA was generated using in-house produced or commercial Tn5 and in a variety of tagmentation buffers and conditions. |
| 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. |
| Experimental Protocol | cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using in-house Tn5 on 0.1pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using in-house Tn5 on 100pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using in-house Tn5 on 10pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using in-house Tn5 on 1pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using in-house Tn5 on 500pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using in-house Tn5 (low) on 0.1pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using in-house Tn5 (low) on 1pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using in-house Tn5 (low) on 500pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using Nextera XT on 0.1pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using Nextera XT on 100pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using Nextera XT on 10pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using Nextera XT on 1pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. cDNA was generated using SMART-seq2 (Picelli et al. Nature Methods 2013); Tagmentation using Nextera XT on 500pg cDNA; cDNA was generated from cells according to Smart-seq2 (Picelli et al. Nature Methods 2013); We prepared sequencing libraries from cDNA using in-house or commercial Tn5 using tagmentation conditions as outlined in the associated study. |
| Data processing | All reads were aligned to the human (hg19) or mouse (mm10) genomes using STAR (Dobin et al. 2013); Non-uniquely mapped reads were removed; Rpkmforgenes (available at http://sandberg.cmb.ki.se/rnaseq/), options -readcount -fulltranscript -mRNAnorm -rmnameoverlap -u, was used to generate RPKM values and read counts. We used a file with unique positions from Storvall et al. 2013 PLOS ONE. Gene annotations used were transcripts in RefSeq (Feb 2013).; Genome_build: hg19 and mm10; Supplementary_files_format_and_content: RPKM gene expression values (tab-delimited text) |
| Platform | GPL11154;GPL13112;GPL15228 |
| Public On | Public on Jul 14 2014 |