GSM7102984: OHS s3atac 1 (s3 O1); Homo sapiens; ATAC-seq
The PBMC pellet was thawed and suspended in NIB-HEPES prior to Dounce homogenization. s3-ATAC was then performed as described previously (Mulqueen et al. 2021). Briefly, nuclei were flow sorted via a Sony SH800 to remove debris and attain an accurate count per well before PCR in 1x TD buffer. Immediately following sorting completion, the plate was sealed and spun down for 5 min at 500 r.c.f. and 4  °C to ensure nuclei were within the buffer. Nucleosomes and remaining transposases were then denatured with the addition 1 µL of 0.1% SDS (roughly 0.01% final concentration) per well. Then 4 µL of NPM (Nextera XT Kit, Illumina Inc.) per well was subsequently added to perform gap-fill on tagmented gDNA, with an incubation at 72  °C for 10 min. Next, 1.5 µL of 1 µM A14-LNA-ME oligo was then added to supply the template for adapter switching. The polymerase-based adapter switching was then performed with the following conditions: initial denaturation at 98  °C for 30 s, ten cycles of 98  °C for 10 s, 59  °C for 20 s and 72  °C for 10 s. The plate was then held at 10  °C. After adapter switching 1% (v/v) Triton-X 100 in ultrapure H2O (Sigma 93426) was added to quench persisting SDS. The following was then combined per well for PCR: 16.5 µL of sample, 2.5 µL of indexed i7 primer at 10 µM, 2.5 µL of indexed i5 primer at 10 µM, 3 µL of ultrapure H2O, 25 µL of NEBNext Q5U 2× Master mix (New England Biolabs M0597S) and 0.5 µL of 100× SYBR Green I (Thermo Scientific S7563) for a 50 µL of reaction per well. A real-time PCR was performed on a Bio-Rad CFX with the following conditions, measuring SYBR fluorescence every cycle: 98  °C for 30 s; 16–18 cycles of 98  °C for 10 s, 55  °C for 20 s, 72  °C for 30 s, fluorescent reading, 72  °C for 10 s. After fluorescence passes an exponential growth and begins to inflect, the samples were held at 72  °C for another 30 s then stored at 4  °C. Amplified libraries were then cleaned by pooling 25 µL per well into a 15 mL conical tube and cleaned via a Qiaquick PCR purification column following the manufacturer's protocol (Qiagen 28106). The pooled sample was eluted in 50 µL 10 mM Tris-HCl, pH 8.0. Library molecules then went through a size selection via SPRI selection beads (Mag-Bind TotalPure NGS Omega Biotek M1378-01). Next, 50 µL of vortexed and fully suspended room temperature SPRI beads were combined with the 50-µL library (1× clean up) and incubated at room temperature for 5 min. The reaction was then placed on a magnetic rack and once cleared, the supernatant was removed. The remaining pellet was rinsed twice with 100 µL of fresh 80% ethanol. After ethanol was pipetted out, the tube was spun down and placed back on the magnetic rack to remove any lingering ethanol. Next, 31 µL of 10 mM Tris-HCl, pH 8.0 were then used to resuspend the beads off the magnetic rack and allowed to incubate for 5 min at room temperature. The tube was again placed on the magnetic rack and once cleared, the full volume of supernatant was moved to a clean tube. DNA was then quantified by Qubit double-stranded DNA High-sensitivity assay following the manufacturer's instructions (Thermo Fisher Q32851). Libraries were then diluted to 2 ng µL−1 and run on an Agilent Tapestation 4150 D5000 tape (Agilent 5067-5592). Library molecule concentration within the range of 100–1,000 base pairs (bp) was then used for final dilution of the library to 1 nM.