GSM6944029: ATAC-seq for iPSCs-derived motor neurons from a healthy people_1; Homo sapiens; ATAC-seq
Human B lymphocytes (~1×105) or progenitor-differentiated motor neurons (~1.6×106) were used for ATAC-seq. Cells were pelleted in 1× PBS (500 g for 5 min, 4°C) and resuspended in 50 μl of cold lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAL CA-630) with gentle pipetting. Cell lysates were centrifuged (500×g for 10 min, 4°C) to pellet the nuclei, which were then incubated with 50 μl of transposition reaction buffer (25 μl of 2× Tagment DNA reaction buffer [Illumina FC-121-1030], 2.5 μl of Nextera Tn5 Transposase [Illumina FC-121-1030], and 22.5 μl of nuclease-free H2O) at 37°C for 30 min with gentle rotation. After the reaction, genomic DNA was purified with a Qiagen MinElute PCR Purification Kit (Qiagen, 28204) and eluted in 10 μl of elution buffer (10 mM Tris buffer [pH 8.0]). To amplify the transposed DNA fragments, the DNA sample was mixed with PCR amplification buffer containing nuclease-free H2O, Nextera PCR Primer 1, Nextera PCR Primer 2 (barcode), and NEBNext High-Fidelity 2× PCR Master Mix (New England Labs, M0541), and then amplified by 1 cycle of 72°C incubation for 5 min and 98°C for 30 sec, followed by 5 cycles of 98°C for 10 sec, 63°C for 30 sec, and 72°C for 1 min. To avoid PCR saturation of a library and reduce the GC and size bias, 5 μl of the first PCR reaction were amplified in a side qPCR reaction to select an appropriate cycle number for the second-round PCR reaction, which was set to 1 cycle of 98°C incubation for 30 sec, followed by 20 cycles of 98°C for 10 sec, 63°C for 30 sec, and 72°C for 1 min. The cycle number corresponding to a quarter of the maximum fluorescence intensity was used in the final PCR amplification. After the final amplification, PCR products were purified using a Qiagen MinElute PCR Purification Kit (Cat. NO. 28204) and analyzed by deep sequencing.