GSM7102967: UCS ddseq 1 (ddSEQ U1); Homo sapiens; ATAC-seq
Cryopreserved PBMC cells were quickly thawed in a 37  °C water bath, rinsed with culture medium (RPMI medium supplemented with 15% FBS) and then treated with 0.2 U μl−1 DNase I (Thermo Fisher Scientific) in 5 mL of culture medium at 37 °C for 30 min. After DNase I treatment, cells were washed once with medium and then twice with ice-cold 1× PBS with 0.1% BSA. Cells were then filtered with a 35-μm cell strainer (Corning) and cell viability and concentration were measured with Trypan blue on the TC20 Automated Cell Counter (Bio-Rad). For a detailed description of tagmentation protocols and buffer formulations, refer to the SureCell ATAC-Seq Library Prep Kit User Guide (17004620, Bio-Rad). Collected cells and tagmentation buffers were chilled on ice. Lysis was performed simultaneously with tagmentation. After washing, equal number of cells from each donor were mixed with Whole Cell Tagmentation Mix containing 0.1% Tween-20, 0.01% Digitonin, 1× PBS supplemented with 0.1% BSA, ATAC Tagmentation Buffer and ATAC Tagmentation Enzyme (ATAC Tagmentation Buffer and Enzyme are both included in the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad)). The mix was split into two technical replicates and the cells were then mixed and agitated on a ThermoMixer (5382000023, Eppendorf) for 30 min at 37  °C. Tagmented cells were kept on ice before encapsulation.  Tagmented cells were loaded onto a ddSEQ Single-Cell Isolator (12004336, Bio-Rad). scATAC-seq libraries were prepared using the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad) and SureCell ddSEQ Index Kit (12009360, Bio-Rad). Bead barcoding and sample indexing were performed in a C1000 Touch thermal cycler with a 96-Deep Well Reaction Module (1851197, Bio-Rad); PCR conditions were as follows: 37 °C for 30 min, 85 °C for 10 min, 72 °C for 5 min, 98 °C for 30 s, eight cycles of 98 °C for 10 s, 55 °C for 30 s and 72 °C for 60 s, and a single 72 °C extension for 5 min to finish. Emulsions were broken and products were cleaned up using Ampure XP beads (A63880, Beckman Coulter). Barcoded amplicons were further amplified using a C1000 Touch thermal cycler with a 96-Deep Well Reaction Module; PCR conditions were as follows: 98 °C for 30 s, seven cycles of 98 °C for 10 s, 55 °C for 30 s and 72 °C for 60 s, and a single 72 °C extension for 5 min to finish. PCR products were purified using Ampure XP beads. and quantified on an Agilent Bioanalyzer (G2939BA, Agilent) using the High-Sensitivity DNA kit (5067-4626, Agilent).