title: GSM6426716: ATAC-seq 3; Aedes aegypti; ATAC-seq
library_construction_protocol: Aag2 cells were trypsinized on plates and resuspended into single cells. Cells were pelleted by spinning at 500 × g for 5 min at 4°C and then washed twice with ice-cold 1× PBS buffer. 50,000 cells were counted and suspended in ice-cold 1× PBS. The same volume of 2× lysis buffer (20 mM Tris-HCl, 20 mM NaCl, 30 mM MgCl2, 0.5% IGEPAL) was added to 50,000 cells and pipetted up and down for 20 times to lyse the cells. Cells were pelleted at 500 × g for 30 min at 4°C and suspended in 25 µL of digestion buffer (10 mM Tris pH 7.6, 5 mM MgCl2, 10% DMF, 1U Tn5 transposase), followed by tagmentation in 37°C incubator for 1 h at 650 rpm. After tagmentation, 9 µL of clean-up buffer (0.5 M NaCl, 150 mM EDTA, 1% SDS, 2 mg/mL Protease K) was added for incubation at 40°C for 30 min with 650 rpm shaking. Normal phase 2× AMPure XP beads (68 µL) purification was performed to remove small fragments (<100 bp). Purified DNA was used for a first PCR amplification (Nextera Index Kit) with KAPA HiFi HotStart ReadyMix (Roche) for seven cycles. Reverse phase 0.55× SPRI bead (27.5 µL) purification was performed to remove large fragments (>300 bp), followed by purification with QIAquick PCR Purification Kit (Qiagen). Purified DNA was used for a second PCR amplification (Nextera Index Kit) with KAPA HiFi HotStart ReadyMix (Roche) for another seven cycles. The PCR product was purified again with QIAquick PCR Purification Kit (Qiagen). DNA amounts were determined with Qubit fluorometric quantification (ThermoFisher Scientific) and analyzed for fragment size distribution with a DNA 1000 Chip on a Bioanalyzer 2100 system (Agilent). Prepared libraries can be directly loaded to sequencer