title: ATAC-seq (protocol v2; 100K 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, and carefully layered on top of a sucrose gradient with 0.3M sucrose buffer on top and 1.8M sucrose buffer on the bottom (0.3M or 1.8M D-Sucrose, 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2) and centrifuged (4500 x g for 45 min at 4C). Pelleted nuclei were resuspended in PBS for counting. Nuclei concentration was determined by counting on a hemocytometer, an aliquot of 50,000-500,000 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 10 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. If a library was not concentrated enough for sequencing after 10 cycles of amplification, library complexity was a concern, and library preparation was repeated. Libraries were assessed for fragment length distribution on a Bioanalyzer High Sensitivity DNA Chip (Agilent Genomics), and submitted for paired-end 40 bp sequencing on the NextSeq platform.