Nebnext pcr master mix

ATAC-seq was performed using Nextera DNA library prep kit (Illumina, 15028212) as previously described (Pastor et al., 2018 (link)). Cells were collected in lysis buffer (10mM Tris pH7.4, 10mM NaCl, 3mM MgCl₂, 1% NP40) and spun at 500 g at 4°C for 10 min. The pellet was resuspended in the transposase reaction mix (25 μL 2 × Tagmentation buffer, 2.5 μL transposase and 22.5 μL nuclease-free water) and incubated at 37°C for 30 min. The samples were purified using MinElute PCR Purification Kit (QIAGEN, 28006) and amplified using 1 × NEBnext PCR master mix (NEB, M0541S) and 1.25 μM of custom Nextera PCR primers 1 and 2 with the following PCR conditions: 72°C for 5 min; 98°C for 30 s; and thermocycling at 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. Samples were amplified for five cycles and 5 μL of the PCR reaction was used to determine the required cycles of amplification by real-time PCR. The remaining 45 μL reaction was amplified with the determined cycles and purified with MinElute PCR Purification Kit (QIAGEN, 28006) yielding a final library concentration of about 30 nM in 20 μL. Libraries were subjected to pair-end 50bp sequencing on HiSeq 2000 or HiSeq 2500 sequencer with 4–6 indexed libraries per lane.

ATAC-Seq was performed as described (31 ). Briefly, 50,000 BM CD27⁺CD11b⁻ NK cells from RId2^−/− and Ctrl mice and CD27⁻CD11b⁺ NK cells from Ctrl mice were sorted and used for each ATAC-Seq assay (2 assays). Nuclei were isolated lysing the cells with cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂ and 0.1% IGEPAL CA-630) followed by centrifugation at 500g for 10 min at 4 °C. The supernatant was carefully removed and the pellet was resuspended in transposase reaction mix (25 μl 2× Tagment buffer, 2.5 μl Tagment DNA enzyme (Illumina, FC-121–1030) and 22.5 μl nuclease-free water) and incubated at 37 °C for 30 min. After transposition, the sample was purified with a Qiagen MinElute kit and library fragments were amplified using Nextera PCR Primers (IlluminaNextera Index kit) and NEBnext PCR master mix (New England BioLabs, 0541) for a total of 10–12 cycles followed by purification with a Qiagen PCR cleanup kit.
The amplified, adaptor-ligated libraries were size-selected with Life Technologies E-Gel SizeSelect gel system in the range of 150–650 bp and quantified with an Agilent Bioanalyzer and via qPCR using the KAPA Library Quantification Kit on the Life Technologies Step One System. Libraries were sequenced on an Illumina HiSeq2000 to generate 7.5× 10⁷ to 10 × 10⁷ 50-bp paired-end reads.

For ATAC-seq, 5–10 × 10⁴ YFP⁺CD25⁺ T_reg cells were FACS sorted from MACS-prepurified CD4⁺ T cells (CD4⁺ T Cell Isolation Kit, mouse; 130-104-454, Miltenyi) isolated from lymph nodes of 21-day-old Foxp3^YFP-CreCtnnb1^fl(ex3) and Foxp3^YFP-Cre WT mice. Cells were centrifuged at 500g at 4 °C for 5 min, washed with 1× PBS, and centrifuged again. Cells were resuspended in lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl₂ and 0.1% IGEPAL CA-630) and immediately centrifuged at 500g at 4 °C for 10 min. Pellets were resuspended in transposition reaction buffer (25 μl 2× Tagment Buffer (FC-121-1030, Illumina)), 2.5 μl Tagment DNA Enzyme and 22.5 μl nuclease-free H₂O) for 30 min at 37 °C. DNA was purified with a Qiagen MinElute Kit and amplified with Nextera PCR primers (Illumina Nextera Index Kit) and NEBNext PCR Master Mix (M0541, New England BioLabs). Amplified DNA was purified with a Qiagen PCR cleanup kit. Libraries were sequenced on a HiSeq 4000 sequencer at the University of Chicago Genomics Facility.