title: ATAC-seq (protocol v1; 50K cells) of adult chicken lung
design: One gram of tissue was minced with a razor blade, transferred to a gentleMACS C tube with 10 mL of sucrose buffer (250 mM D-Sucrose, 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2; 1 protease inhibitor tablet per 50 mL solution just prior to use), and homogenized using the gentleMACS Dissociator Program E.01c Tube, twice. Homogenate was filtered using a 100 uM Steriflip Vacuum Filter system. Volume was brought up to 9.9 mL with sucrose buffer, and 1.1 mL DMSO was added to achieve a 10% final concentration. Solution was aliquoted into cryotube vials, frozen overnight at -80C in a Nalgene Cryo 1C/min Freezing Container, and then stored at -80C long-term. Cryopreserved nuclei preparations were thawed on ice, pelleted (600 x g for 10 min at 4C), resuspended in sucrose buffer (250 mM D-Sucrose, 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2), filtered through a 20 m Steriflip Vacuum filter system, pelleted again (500 x g for 5 min at 4C), washed with PBS (500 x g for 5 min at 4C), and resuspended in PBS for counting. Nuclei concentration was determined by counting on a hemocytometer, an aliquot of 50,000 nuclei was pelleted in a 1.5 mL Eppendorf tube (500 x g for 5 min at 4C), and the nuclear pellet was incubated in 50 L transposition mix (25 L TD buffer, 2.5 L TDE1 enzyme, 22.5 L ddH2O) for 30 minutes at 37C (300 rpm). Transposed DNA was then purified with the MinElute PCR Purification kit (DNA was eluted with 10 L Buffer EB). DNA was then amplified by PCR. 40 L PCR master mix (25.4 L SsoFast EvaGreen Supermix, 13 L ddH2O, 0.8 L 25 M Custom Primer 1, 0.8 L 25 M Custom Primer 2) was added to 10 L eluted DNA and cycled as follows: 1 x [5 min at 72C, 30 sec at 98C], then 5 x [10 sec at 98C, 30 sec at 63C, 1 min at 72C]. To determine the optimal number of PCR cycles, 5 l of the PCR reaction was added to 10 l of qPCR master mix (6.45 l SsoFast EvaGreen Supermix, 3.25 l ddH2O, 0.15 l 25 M Custom Primer 1, 0.15 l 25 M Custom Primer 2) and cycled on a qPCR instrument as follows: 1 x [30 sec at 98C], then 20 x [10 sec at 98C, 30 sec at 63C, 1 min at 72C]. Linear fluorescent intensity (Rn) was plotted against cycle to determine the additional number of cycles needed (N): the number of cycles required to reach one third of the maximum Rn. In this case it was determined that 7 additional cycles were needed. The remaining 45 l of the PCR reaction was then cycled as follows: 1 x [30 sec at 98C], then (N=7) x [10 sec at 98C, 30 sec at 63C, 1 min at 72C]. Libraries were then purified with the MinElute PCR Purification kit and eluted in 10 L Buffer EB. Libraries were assessed for fragment length distribution on a Bioanalyzer High Sensitivity DNA Chip (Agilent Genomics) and submitted for paired-end 100 bp sequencing on the HiSeq3000 platform.