D4551 250ml

Tn5 reaction buffer was prepared: 50 mM TAPS (Sigma #T9659-100G), 25 mM MgC12, (Ambion#AM9530G); the pH was adjusted to 8.5 and the solution was sterile filtered. Tn5 dilution buffer was prepared: 50 mM Tris-HC1 pH 7.5 (Sigma #T2944-100ML), 100 mM NaCl (Sigma #S5150-1L), 0.1 mM EDTA (Invitrogen #AM9260G), 50% glycerol (Sigma #G5516-100ML), 0.1% Triton-X100 (Sigma #X100-100ML), and supplemented with fresh 1 mM DTT (Sigma #646563-10x.5ML) before use. To achieve maximum library complexity, the entire sample was processed in several tagmentation reactions with 1 ng input each. cDNA was diluted to 0.2 ng/μl with nuclease-free water and 5 μl (1 ng) per reaction was distributed into a 96-well plate on ice. A mix of 11.25 μl nuclease-free water, 5 μl of 5x Tn5 reaction buffer, 2.5 μl of dimethylformamide (Sigma #D4551-250ML), and 1.25 μl of freshly diluted i7-only transposome (prepared as described below and diluted 1:4.5 in Tn5 dilution buffer) was added. Reactions were incubated for 10 min at 55 °C, then cooled for 1 min on ice. The enzyme was inactivated by addition of 2.5 μl of 1% SDS (Sigma #71736-100ML) for 5 min at room temperature. Next, the volume was brought to 50 μl and the fragmented cDNA was purified with a 1.0x SPRI cleanup, eluting in 17 μl of EB buffer (Qiagen #19086).