GSM6481367: MD6_ATAC_3; Homo sapiens; ATAC-seq
Samples were resuspended in 50uL Tn5 transposase mixture: 1x Tagment DNA Buffer, 0.5uL Tagment DNA Enzyme (Nextera DNA Library Preparation Kit, Illumina). Transposition reaction was incubated at 37°C for 45 minutes, followed by DNA isolation using a Qiagen MinElute PCR purification kit (QIAGEN, Hilden, Germany). Libraries were amplified for 5 cycles using the NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs, MA, USA) with primers (0.5uM each) from (Buenrostro et al., 2015) and the following the following cycling program: 5 minutes at 72°C, 30 s at 98°C, followed by 5 cycles [98°C for 10 s, 63°C for 30 s and 72°C for 60 s] and a final extension at 72°C for 5 minutes. To avoid overamplification of libraries, 2uL of the eluted DNA were subjected to a side-qPCR for defining the optimal number of PCR cycles for each sample.The remaining libraries were subjected to a second round of PCR in a volume of 50uL with NEBNext High-fidelity 2x PCR master mix, respective primers (1.25uM each) and the following thermal cycles: 30 s hot-start at 98°C, followed by 7-13 cycles [98°C for 10 s, 63°C for 30 s and 72°C for 60 s] and a final extension at 72°C for 5 minutes. The final libraries were purified using Agencourt AMPure XP beads, eluted in 30uL Tris HCl 10mM pH 8 and quantified using the 2100 Bioanalyzer (Agilent cat. #G2939BA) and the Qubit fluorometer (Thermo Fisher cat. #Q33226)