title: GSM8281746: HUVEC, ATAC-Seq, 39 °C heat stress for 2 hours, biological replicate 2 (HS2); Homo sapiens; ATAC-seq
library_name: GSM8281746
library_construction_protocol: HUVEC cells were grown in dishes, cryopreserved in 1ml of media with DMSO containing 0.5-1.5x10^6 cells. The experiment was done in biological duplicate, paired with RNA-seq experiment. The ATAC-seq experiments have been conducted by the Diagenode ATAC-seq Profiling service (Diagenode Cat# G02060000).  Transposition. Cell viability and cell counting were evaluated by visualization under the microscope (AE2000; Matic). 200,000 cells were centrifuged at 500g before proceeding with the nuclei isolation and transposition reaction using the ATAC-seq Kit from Diagenode (Diagenode Cat #C01080002). According to the results of the nuclei isolation optimization, the Tween20/Igepal concentration in Lysis buffer 1 used was 0,1% with an incubation time of 3 minutes. 50,000 counted nuclei were transferred to a new tube and were centrifuged at 500g before proceeding with the transposition reaction. Isolated nuclei were lysed and transposed for 30 minutes at 37°C using the prokaryotic Tn5 transposase system (Nextera DNA library kit, Illumina, FC-121–1030). Transposed DNA was then purified on Diapure columns (Diagenode Cat# C03040001).  Transposition. Cell viability and cell counting were evaluated by visualization under the microscope (AE2000; Matic). 200,000 cells were centrifuged at 500g before proceeding with the nuclei isolation and transposition reaction using the ATAC-seq Kit from Diagenode (Diagenode Cat #C01080002). According to the results of the nuclei isolation optimization, the Tween20/Igepal concentration in Lysis buffer 1 used was 0,1% with an incubation time of 3 minutes. 50,000 counted nuclei were transferred to a new tube and were centrifuged at 500g before proceeding with the transposition reaction. Isolated nuclei were lysed and transposed for 30 minutes at 37°C using the prokaryotic Tn5 transposase system (Nextera DNA library kit, Illumina, FC-121–1030). Transposed DNA was then purified on Diapure columns (Diagenode Cat# C03040001). Library preparation. Libraries were prepared from purified transposed DNA using NEBNext High-Fidelity PCR MasterMix (NEB, M0541) and Illumina indexing primers. The number of library amplification cycles was determined by qPCR analysis using NEBNext High-Fidelity PCR MasterMix (NEB, M0541) on LightCycler® 96 System (Roche). After their amplification, the libraries were size selected and purified using Agencourt® AMPure® XP (Beckman Coulter) and quantified using Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32854). Finally, their fragment size was analyzed by High Sensitivity NGS Fragment Analysis Kit (DNF-474) on a Fragment Analyzer™ (Advanced Analytical Technologies, Inc.). Libraries were pooled and sequenced on an Illumina NovaSeq6000 in a 2 × 50-bp, paired-end mode.