title: GSM4634705: SPT6i_ATACseq_rep1; Homo sapiens; ATAC-seq
library_construction_protocol: Cells were washed briefly with phosphate-buffered saline (PBS) two times. Permeabilized nuclei were obtained by resuspending cells in 250µL Nuclear Permeabilization Buffer [(10 mM Tris-HCl (pH 7.4) (BP-152, Thermo Fisher Scientific), 10 mM NaCl (S271-3, Thermo Fisher Scientific), 3 mM MgCl2 (194698, MP Biomedicals), 0.1% Tween-20 (P7949, Sigma), 0.1% IGEPAL-CA630 (I8896, Sigma), 0.01% digitonin (G9441, Promega) in Molecular biology water (46000-CM, Corning)], and incubating for 5 min on a rotator at 4°C. Nuclei were then pelleted by centrifugation for 5 min at 500 x g 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 10 µL ice-cold Tagmentation Buffer, and incubated with 1µL 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. Sequencing was performed at the UC San Diego IGM Genomics Center on an Illumina NovaSeq 6000 using the 100 bp paired-end protocol. The ATAC-seq was performed in CTLi and SPT6i cells in duplicates.
attributes.tag: GEO Accession
attributes.value: GSM4634705