GSM3895496: ATAC-seq 1R Rep #2; Arabidopsis thaliana; ATAC-seq
Nuclei were purified by INTACT, transposition was performed by incubation with Tn5, and DNA was extracted by use of Qiagen Minelute ATAC-seq library preparation was performed according to (Buenrostro et al., 2013).  Briefly, a reaction was prepared to amplify the sample in a two step process. The reaction was assembled containing transposed DNA (10 µL), 5 µM Ad1.1 and an indexing primer (Buenrostro, Giresi, Zaba, Chang, & Greenleaf, 2013), 1X NEBNext High Fidelity PCR mix and cycled in the following program: 72oC 5 min; 98oC 30 s; 3 cycles of 98oC 10 s, 63oC 30s, 72oC 1 min; 4oC and the samples were placed on ice.  Then a qPCR was performed using an aliquot of the amplified library with the following program: 98oC for 30 s; 20 cycles of 98oC for 10 s, 63oC for 30 s, 72oC for 1 min.  The original PCR reaction was resumed for n additional cycles, where n is the cycle at which the qPCR reaction was at 1/3 of its maximum fluorescence intensity.  Amplified DNA was then purified with Qiagen MinElute PCR purification columns and eluted in 20 µL of EB. Amplified libraries were purified and size selected for fragments between 200 to 600 bp using SPRI AMPure XP beads (Beckman).  AMPure XP beads were added at a 0.55:1.0 bead to sample ratio, and the remaining DNA was recovered by the addition of AMPure XP beads at a 1.55:1.0 bead to sample ratio and eluted in 13 µL of EB. Library concentration was determined using the NEBNext (New England Biolabs) library quantification kit.  Final libraries were multiplexed to >5 nM final concentration and sequenced on the HiSeq 3000/4000