OpenTn5: Open-Source Resource for Robust and Scalable Tn5 Transposase Purification and Characterization [sciATAC-Seq]

Tagmentation is an efficient approach for sequencing library preparation, which combines DNA fragmentation by transposition of sequencing adapters, using the bacterial cut-and-paste transposase Tn5. Here we present an open-source protocol for the generation of multi-purpose hyperactive Tn5 transposase, including its benchmarking in CUT&Tag, bulk and single-cell ATAC-seq. The OpenTn5 protocol yields multi-milligram quantities of pG-Tn5E54K, L372P protein per liter of E. coli culture, sufficient for thousands of tagmentation reactions and the enzyme retains activity in storage for more than a year. Overall design: To determine if the performance of in-house pG-Tn5 is comparable to commercial pAG-Tn5 from Epicypher (SKU: 15-1017), we performed CUT&Tag using both enzymes to profile the distribution of H3K27me3 and H3K27ac histone modifications in MCF7 cells. To compare the performance of pG-Tn5 with commercially available Tn5 (Illumina TDE1, CAT: #20034198), we conducted ATAC-seq experiments in MCF7 and K562 human cell lines with matched enzyme volumes. We also performed an in-house pG-Tn5 transposome titration in K562 cells to understand how varying pG-Tn5 enzyme concentration influences ATAC-seq signal globally. We also demonstrated pG-Tn5 can be readily applied to sensitive emerging applications that require custom adapter loading, such as EasySci-ATAC, which was performed in MCF7 cells.