title: GSM3595722: SS_AT_MC_2uM-Cis-1_5; Mus musculus; ATAC-seq
library_construction_protocol: Permeabilized nuclei were obtained by resuspending cells in 250µL Nuclear Permeabilization Buffer [0.2% IGEPAL-CA630 (I8896, Sigma), 1mM DTT (D9779, Sigma), Protease inhibitor (05056489001, Roche), 5% BSA (A7906, Sigma) in PBS (10010-23, Thermo Fisher Scientific)], and incubating for 10 min on a rotator at 4°C. Nuclei were then pelleted by centrifugation for 5 min at 500 xg at 4°C. The pellet was resuspended in 25µL ice-cold Tagmentation Buffer [33 mM Tris-acetate (pH = 7.8) (BP-152, Thermo Fisher Scientific), 66 mM K-acetate (P5708, Sigma), 11 mM Mg-acetate (M2545, Sigma), 16 % DMF (DX1730, EMD Millipore) in Molecular biology water (46000-CM, Corning)]. An aliquot was then taken and counted by hemocytometer to determine nuclei concentration. Approximately 50,000 nuclei were resuspended in 20µL ice-cold Tagmentation Buffer, and incubated with 1mL Tagmentation enzyme (FC-121-1030; Illumina) at 37 °C for 30 min with shaking 500 rpm. The tagmentated DNA was purified using MinElute PCR purification kit (28004, Qiagen). The libraries were amplified using NEBNext High-Fidelity 2X PCR Master Mix (M0541, NEB) with primer extension at 72°C for 5 min, denaturation at 98°C for 30s, followed by 8 cycles of denaturation at 98°C for 10 s, annealing at 63°C for 30 s and extension at 72°C for 60 s. Amplified libraries were then purified using MinElute PCR purification kit (28004, Qiagen), and two size selection steps were performed using SPRIselect bead (B23317, Beckman Coulter) at 0.55X and 1.5X bead-to-sample volume rations, respectively. Each library was then sequenced on an Illumina NextSeq500 or HiSeq4000 to a depth of >= 25 million usable reads pairs (i.e. after mapping, filtering, and elimination of PCR duplicates as described in the data processing section).
attributes.tag: GEO Accession
attributes.value: GSM3595722