Nextera dna library prep kit

ATAC-seq was performed as previously reported (Buenrostro et al., 2013 (link)), with a few modifications. Briefly, 5 × 10⁴ cells were FACS sorted, washed once with ice-cold PBS, and lysed in 300 µl lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl₂, and 0.1% NP-40) by gently pipetting up and down. After centrifugation, the supernatant was removed. Then, 50 µl of reaction mix containing 25 µl TD Buffer, 2.5 µl TDE1 (Illumina Nextera DNA Library Prep Kit), and 22.5 µl nuclease-free water was immediately added to set up a transposition reaction at 42°C for 40 min. DNA was immediately purified afterward by using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). The transposed DNA was amplified by PCR for 10–12 cycles with the Nextera DNA Library Prep Kit and Nextera XT Indexing Kit (Illumina). The library DNA within the 150–500-bp range was enriched by one round of negative selection with 0.6 volume AMPure XP beads (Beckman Coulter) and two rounds of positive selection with 1 volume AMPure XP beads. The libraries were quantified by NEBNext Library Quant Kit for Illumina (New England Biolabs) and sequenced, with paired-end 100-cycle sequencing performed on a HiSeq 4000 or HiSeq 2500 sequencer (Illumina).

After neutralization, 39 nL of PCR master mix (19.5 nL NPM (Nextera DNA Library Prep Kit, Illumina), 6.5 nL PPC (Nextera DNA Library Prep Kit, Illumina), 0.65 nL 10% Tween-20, 12.35 nL PCR water) was dispensed to each well. PCR was performed using the following conditions: 72°C for 3 min; 95°C for 30 s; 8 cycles or 11 cycles of 95°C for 10 s, 55°C for 30 s and 72°C for 30 s; 72°C for 3 min; and finally 10°C.