GSM6454206: ATACSeq_DIPG4_with_DSMO_Radiation 2; Homo sapiens; ATAC-seq
100,000 cells were spined down at 500 xg, 5min at 4oC, pellet was washed once with 500ul of cold 1x PBS, and spined down at 500 xg, 5min at 4oC. Pellet was resuspended in 450ul of cold hypotonic buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2) and immediately added 50ul of 1% IGEPAL CA-630 (0.1% final) and inverted to mix. Cells were incubated on ice for 15 min, spined down at 500 xg for 10 min at 4oC. Pellet was set on ice and the transposition reaction was performed following the ATAC-seq Kit instructions (Cell Biologics, Cat No. CB6936). Pellet cells were resuspended in 50ul of transposition reaction mix (25ul 2X Reaction Buffer, 2.5ul Transposome, 22.5ul Nuclease free water) and incubated at 37oC for 1 hour. DNA was purified using Qiagen MinElute kit (Qiagen, #28004) and eluted in 15ul of Elution Buffer, followed by the Library Generation to amplify transposed DNA fragments. 10ul of Transposed DNA, 10ul of Nuclease free water, 2.5ul of Ad1.noMX (Oligo 1), 2.5 ul Ad2._Barcode (Oligo 2) and 25ul of High Fidelity 2x PCR Master Mix and ran on a thermocycler using the following cycle: 98oC 30sec, (98oC 10sec, 63oC 30sec, 72oC 1min) x 10 times and hold at 4oC.  Followed by the Library Purification. For double-sided bead purification (to remove primer dimers and large >1,000 bp fragments), each PCR sample was transferred to a 1.5ml tube and 0.5X volume (25ul) of AMPure XP beads was added, mixed by pipetting 10 times and incubated at room temperature for 10min. Tubes were placed in a magnetic rack for 5min and the supernatant was transferred to a new tube. 1.3X of the original volume (65ul) of AMPure XP beads was added, mixed by pipetting 10x and incubated at room temperature for 10 min. Tubes were placed in a magnetic rack for 5min, supernatant was discarded, and beads were washed with 200ul 80% EtOH. EtOH was removed and tubes were left on magnetic rack with cap open for 10 min or until the pellet was totally dry. Beads were resuspended in 20ul of nuclease-free water and mixed thoroughly, tubes were placed in the magnetic rack for 5min and the supernatant was transferred to a new tube.