title: GSM7114660: KOLF2.1J_NGN2_rep3 [ATAC-seq]; Homo sapiens; ATAC-seq
library_construction_protocol: We harvested 100,000 cells by mechanical dissociation and washed with 50 μL cold 1X PBS and centrifuged at 300xg for 5 minutes. The cell pellet was then resuspended in 50 μL cold Lysis Buffer (10 mM Tris-HCl at pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin, and nuclease-free H2O) and incubated on ice for 3 minutes. After incubation, 1 mL of Wash Buffer (10 mM Tris-HCl at pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, and nuclease-free H2O) was added, samples were inverted gently to mix, and centrifuged at 500xg for 10 minutes at 4°C. The supernatant was discarded and the remaining nuclei were resuspended in 50 μL Transposition Mix (2X TD Buffer, TDE1, and nuclease-free H2O) and incubated at 37°C on shaker at 1000 rpm for 30 minutes. Samples were immediately purified using the Qiagen MinElute Reaction Cleanup kit and eluted in a final volume of 11 μL. Recovered DNA was then used to generate sequencing libraries using primers from the Nextera XT Index Kit (15055293) and Q5 Hot Start Master Mix and amplified (30 sec at 98°C; [10 sec at 98°C, 30 sec at 63°C, and 72°C for 1 min] x10 cycles). Libraries were quantified with Qubit dsDNA HS Assay kit and visualized with BioAnalyzer High Sensitivity DNA Analysis kit (Agilent 5067-4626) and 2100 BioAnalyzer Instrument (Agilent).