Long-read analysis of Tn5 insertion patterns
Long-read analysis of Tn5 insertion patterns
ATAC-seq (30) was modified to enable long-read analysis. Briefly, CCRF-CEM and Jurkat cells were grown overnight, and 100,000 cells were were harvested at 500 × g 4 °C for 5 min. Cell pellets were resuspended in 50 μL of lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween, 0.01% Digitonin) and kept for 4 min on ice, after what 500 μL of wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween) was added and gently resuspended by pipetting and centrifuged at 500 × g 4 °C for 15 min. The nuclei pellets were resuspended in the transposition reaction mix (TD buffer with 0.1 % Tween, 0.01% Digitonin, containing Tn5 transposase). The final concentrations of Tn5 in the reaction were 50 ng/µl. Reactions were incubated for 2 h at 37 °C shaking at 700 rpm.  CCRF-CEM (CCL-119, ATCC) and Jurkat cells, both of T cell origin, were cultured in RPMI 1640 medium (cat no. 21870076, Thermo Fisher Scientific) with 10% Bovine Serum (cat no. A3294-10G, Sigma Aldrich) 1% 100× penicillin-streptomycin-glutamine. For transpososome assembly the preanealed cargo 250 nM duplex PAGE purified oligos were ordered from IDT (Coralville): Sense (5'-CTGTCTCTTATACACATCTACGCGGTGGACAAAAAATTTCATTTGGAACTAGATTTGACCTCAGCTTCAATGCCAGAGATGTGTATAAGAGACAG) and antisense (5'-CTGTCTCTTATACACATCTCTGGCATTGAAGCTGAGGTCAAATCTAGTTCCAAATGAAATTTTTTGTCCACCGCGTAGATGTGTATAAGAGACAG). Cargo oligos of 95bp were created with Mosaic ends (ME) (26) and with a restriction site for BbvCI. Such length was previously found in one of the potential optimal windows for binding Tn5 transposase (27). The body of the cargo was modified from the parts of Tn5 transposon (GenBank KF813062.1) that were not creating the hairpin according to the folding tool Mfold (28). The cargo concentration was adjusted to 67 µM in the buffer (2mM DTT, 771mM NaCl, 44.7mM Tris-HCl pH 7.5, 10% glycerol) and stored at -20°C. Tn5 E54K,L372P manufactured and purified at EMBL Heidelberg (29), was removed from -80°C and thawed. Glycerol concentration was adjusted to 50% and stored for a few weeks at -20°C. Tn5 was assembled with cargo oligonucleotides at equal molar. The mixture was incubated at room temperature, shaking at 400 revolutions per minute (rpm) for 1 h. The transposed gDNA was purified using the Monarch Genomic DNA Purification Kit (catalog no. T3010S, New England Boilabs) and length and quality were accessed by gel. The libraries were prepared by SciLifeLab