Mua transposase

ATAC-seq was performed as described [39 (link)], with the major exception of the use of a MuA transposase (Thermo) rather than the TN5 transposase. Briefly, MCF7 or LOXIMVI cells were trypsinized and 50 K cells, spun down, washed once with PBS, and lysed with a hypotonic buffer containing 0.1% NP-40, and spun down to generate a crude nuclei pellet. This pellet was transposed in a 30 μl volume using MuA (0.7 μl), MuA buffer (10 μl), and H2O (19 μl) for 5 min at 30C. The sample was treated with 3 μl stop solution, and incubated at 30C for a further minute. The sample was then collected and purified by addition of 45 μl of SPRI beads (Aline Biosciences). The purified sample was PCR amplified in two steps to add barcoded adaptors suitable for Illumina sequencing. Samples were sequenced with single end 75 bp reads on an Illumina NextSeq. Reads (> 30 M) were trimmed to remove adaptors with cutadapt [38 ], aligned to the genome with Bowtie, and analyzed with Matlab. Genomic DNA (50 ng) from MCF7 and LOXIMVI was transposed, amplified and sequenced in parallel to estimate background.

Integration site analysis was conducted using MuA-mediated integration site recovery as previously described.⁴³ (link) gDNA was incubated with MuA transposase (Thermo Fisher Scientific) and commercially synthesized annealed oligonucleotides (Data S1, sequence A and B).
MuA digested gDNA was amplified by PCR with a forward primer binding to either super-piggyBac or piggyBat transposon 3′-TIR (Data S1, sequence E), with the generated PCR amplicon library consisting of oligonucleotides spanning the intersection of the 3′ transposon-specific TIR and adjacent gDNA corresponding to genomic integration site. This PCR amplicon library was indexed using Nextera XT Indexing kit (Illumina, San Diego, CA) and purified using JetSeq beads (Bioline, Memphis, TN).
Next-generation sequencing of purified PCR amplicon libraries was performed on the Illumina MiSeq v.3 platform (Australian Genome Research Facility) and mapped to chromosomal loci using previously described bioinformatic algorithms.⁴⁴ (link) Mapped loci were annotated using HOMER hg19 peak enrichment tool,⁴⁵ (link) with frequency of integrations expressed as a log fold change over expected for random insertion. Raw sequencing data have been deposited in the Sequence Read Archive (Sequence Read Archive: PRJNA807916), with all unique identified integration sites provided in Table S1.

FastDigest restriction endonucleases, MuA transposase and T4 DNA ligase were purchased from Thermo Fisher Scientific. DNA Polymerase I, Large (Klenow) Fragment, was purchased from New England Biolabs. All DNA modifying enzymes were used according to the manufacturer’s conditions. All DNA purification procedures were performed according to the manufacturers’ instructions using kits, including GeneJET Plasmid Miniprep kit (Thermo Fischer Scientific) for plasmid extractions from E. coli cells, Zymoclean Gel DNA Recovery kit (Zymo Research) for agarose gel extraction of DNA fragments upon electrophoresis and DNA Clean & Concentrator kit (Zymo Research) for DNA purification and concentration. Bacterial transformations were performed by electroporation using E. cloni 10G ELITE electrocompetent cells (Lucigen).