Minelute reaction cleanup kit

ATAC-seq was performed as described in the Omni-ATAC protocol with minor modifications (51 (link)). A total of 50,000 sorted cardiac endothelial cells were used as an input for the transposase reaction. Reactions were cleaned up with the QIAGEN MinElute Reaction Cleanup Kit (QIAGEN). Libraries were generated and amplified using NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs). Two-sided size selection was carried out using AMPure XP Beads (Beckman Coulter, 1.8× beads).

The Tn5 transposition reaction was performed using the Illumina Tagment DNA TDE1 Enzyme and Buffer Kit (Illumina). For each sample 100,000 cells were collected and washed with cold PBS. A resuspension buffer (10 nM Tris-HCl, pH7.5, 10 mM NaCl, 3 mM MgCl₂) was used for subsequent steps. Cells were lysed in 100 μl lysis buffer (0.1% NP-40, 0.1% Tween-20, and 0.01% Digitonin in resuspension buffer) on ice for 3 minutes, then washed with 1 ml wash buffer (0.1% Tween-20 in resuspension buffer). Lysates were cleared by centrifugation at 500g, 4°C for 10 minutes. The nuclear pellet was resuspended in 100 μl transposition reaction mix (50 μl Tagment DNA Buffer, 33 μl PBS, 1 μl 10% Tween-20, 1 μl 1% Digitonin, 5 μl TDE1, 10 μl nuclease-free water) and incubated at 37°C on a thermomixer with 1,000 rpm for 30 minutes. DNA was isolated using the Qiagen MinElute Reaction Cleanup Kit (Qiagen). Library was prepared with NEBNext High-Fidelity 2X PCR Master Mix (NEB) and SYBR Green I (ThermoFisher), then QC analyzed by the Epigenomics Profiling Core at MD Anderson Cancer Center. Sequencing was performed at the Advanced Technology Genomics Core at MD Anderson Cancer Center using Illumina NovaSeq6000 S1-100 flow cell and 50bp paired-end reading.

Cells were washed with ice-cold FACS buffer and were kept on ice until cell sorting. Twenty-five thousand live cells from each condition were sorted into FACS buffer and were pelleted by centrifugation at 500 × g for 5 min at 4 °C in a precooled fixed-angle centrifuge. Cell lines then were tagmented according to the previously described Fast-ATAC protocol (92 (link)). Briefly, all supernatant was removed with care taken not to disturb the not-visible cell pellet. Transposase mixture (50 μL: 25 μL of 2× TD, 2.5 μL of TDE1, 0.5 μL of 1% digitonin, 22 μL of nuclease-free water) (catalog no. FC-121-1030, Illumina; catalog no. G9441, Promega) was added to the cells, and the pellet was dissociated by pipetting. Transposition reactions were incubated at 37 °C for 30 min in an Eppendorf ThermoMixer with agitation at 300 rpm. Transposed DNA was purified using a Qiagen MinElute Reaction Cleanup kit (catalog no. 28204), and purified DNA was eluted in 12 μL of elution buffer (10 mM Tris⋅HCl, pH 8). Transposed fragments were amplified and purified as described previously (93 (link)) with modified primers (94 ). Libraries were quantified using qPCR before sequencing. All Fast-ATAC libraries were sequenced using paired-end, dual-index sequencing on a NextSeq sequencer (Illumina) with 76 × 8 × 8 × 76 cycle reads at an average read depth of 30 million reads per sample.

For elimination of linear DNA and enrichment of eccDNA, 25 ng of plasma DNA were treated with 5 units of exonuclease V (New England Biolabs) in a 50-µL reaction system at 37 °C for 5 min, followed by column purification using MinElute Reaction Cleanup Kit (Qiagen). For restriction enzyme-based approach, the resultant circular DNA were digested with MspI (New England Biolabs) according to manufacturer’s instructions. The sequencing libraries of the MspI-digested DNA and linear DNA (30 ng of plasma DNA from each case) were prepared using TruSeq Nano DNA LT Library Prep Kit (Illumina). For tagmentation-based approach, circular DNA enriched from 25 ng of plasma DNA were processed using Nextera XT DNA Library Preparation Kit (Illumina). DNA libraries were sequenced on HiSeq 1500/2500 platforms in Rapid Run mode. All libraries were sequenced as 2 × 250-bp paired-end reads.