Instrument: Illumina HiSeq 3000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: HEK293T cells were washed with PBS and permeabilized using a 10 min incubation on ice in 500 mL of complex formation buffer per ~1 million cells. After permeabilization, all subsequent centrifugations of cells were performed using the following procedure: 1 min spin at 250 *g, rotate tubes 180°, repeat spin, leave ~10 mL of solution in the tube when removing buffer. Pelleted cells were washed once with 500 mL of complex formation buffer and suspended in another 500 mL of buffer. Aliquots of 1,000 cell equivalents were transferred volumetrically to clean microcentrifuge tubes and adjusted to 50 mL total volume using complex formation buffer. 5 mL of antibody-bound PA-Tnp complex was added to each 50 mL cell aliquot and incubated at room temperature for 60 min to allow chromatin binding. Unbound complex was removed using three washes performed as follows: pellet and suspend cells in 500 mL of wash buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% Triton X-100), rotate tube for 8 min at room temperature, repeat. Pelleted cells were rinsed with 500 mL of rinse buffer (50 mM Tris pH 8.0, 50 mM NaCl, 0.1% Triton X-100). The rinse buffer was removed and 90 mL of reaction buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM MgCl2, 0.1% Triton X-100) was added to the pelleted cells for a total volume of ~100 mL. The tubes were incubated for 30 min at 37 °C to allow transposition to occur. The reaction was terminated by addition of 4 mL of 0.5 M EDTA, 2 mL of 10% SDS, and 1 mL of 20 mg/mL Proteinase K followed by a 60 min incubation at 65 °C. DNA was purified using phenol-chloroform-isopentanol extraction and ethanol precipitation. The DNA pellet was suspended in 10 mL of 10 mM Tris pH 8.0. PCR reactions for library preparation were performed by adding the following to the 10 mL DNA sample: 25 mL of Phusion High-fidelity PCR Master Mix (NEB catalog # M0531S), 1 mL of 5¢ library index barcode, 1 mL of 3¢ library index barcode, and 13 mL of nuclease-free water. Amplification was performed using an initial step of 72 °C for 5 min followed by 15 cycles of: 98 °C for 10 s, 65 °C for 30 s, 72 °C for 15 s, and a final extension step of 72 °C for 5 min. The PCR products were analyzed using agarose gel electrophoresis. Fragments of the desired size were excised and purified using a QIAquick Gel Extraction kit (Qiagen cat # 28506). scACT-seq