GSM8940045: HO179; Homo sapiens; ATAC-seq
100,000 cells were collected in a 1.5 mL low-binding microcentrifuge tube and brought to a total volume of 1 mL in ice-cold PBS and mixed gently. Cells were pelleted and resuspended in 100 µL of ice-cold anti-freezing buffer (10% DMSO, 10% FBS in DMEM) and mixed gently. Approximately 50 – 100K cells were subjected to ATAC following the Omni-ATAC protocol. Libraries were purified by AMPure XP magnetic beads (Beckman Coulter, A63881) and the size distribution of library DNA was determined by Fragment Analyzer (Advanced Analytical Technologies) using a High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies, DNF-486). Real-time PCR analysis was performed using SYBR Green universal PCR mixes (Bio-Rad) to measure enrichment of accessible regions in the library DNAs. The following primers were used: for accessible regions, Fam86fp-F: 5'-GGCATTAGGGCGGGGATAAA-3', Fam86fp-R: 5'-TAAGACCTGTCTCCTGGGCT-3'; Ccdc146-F: 5'-GCTCCGCCTCTTAGAAACCA-3', Ccdc146-R: 5'-GCGATTCTAACACCTGCCCT-3'; for inaccessible region, hATAC-C19 inter-F: 5'-GCTTGTCTTTCCCAAGTTTACTC-3', hATAC-C19 inter-R: 5'-TAGCTGTCGCACTTCAGAGGA-3'. The libraries were sequenced to 51 base pairs from both ends on an Illumina NovaSeq 6000 SP platform. The concentration and fragmentation profile of submitted ATAC libraries were determined using Qubit fluorometry (Invitrogen) and the Agilent Bioanalyzer. The libraries were sequenced using Illumina's standard protocol for the NovaSeq 6000. The flow cells were sequenced as 50 X 2 paired end reads using the NovaSeq SP sequencing kit and NovaSeq Control Software v1.8.0. Base-calling was performed using Illumina's RTA version 3.4.4.