GSM6738574: rpoa-2::degron; eft-3p::tir-1, 1 mM auxin, replicate 1; Caenorhabditis elegans; ATAC-seq
Synchronized larvae were collected in 50 mM NaCl. Bacteria were cleaned from worms by sedimentation through 5 % sucrose in 50 mM NaCl at 400g for 1 minute with subsequent washes in 50 mM NaCl. At least 40,000 larvae were collected for each condition. Batches of larvae were evenly split into four replicates and buffer was exchanged to Nuclei Wash Buffer (NWB): 10 mMTris-HCl pH 7.5, 40 mM NaCl, 90 mM KCl, 3 mM MgCl2, 0.2 mM DTT, 0.5 mM Spermidine, 0.25 mM Spermine, and 1 X Protease Inhibitor Cocktail. Larvae pelleted in NWB were snap frozen in liquid nitrogen and stored at -70°C.  In order to minimize the potential confounding effects of batch on the experimental design, one replicate of each of the strains were processed concurrently for four total batches of four replicates. To isolate nuclei from larvae, worm pellets were thawed on ice. Buffer was transferred for thawed larvae to Nuclei Prep Buffer (NPB): 10 mMTris-HCl pH 7.5, 40 mM NaCl, 90 mM KCl, 3 mM MgCl2, 0.2 mM DTT, 0.5 mM Spermidine, 0.25 mM Spermine, 0.1 % Triton X-100, 0.1 % IGEPAL CA-630, and 1 X Protease Inhibitor Cocktail. Worms were transferred to a 2 mL dounce homogenizer that was pre-chilled in the refrigerator and kept on ice. Worms were initially broken by 15 strokes with homogenizer pestle A, a short rest on ice, and 15 more strokes with pestle A. Nuclei were released from broken worms by 25 strokes with pestle B, a short rest on ice, and 25 more strokes with pestle B. Debris was pelleted by a low speed spin of the homogenate for 5 minutes at 100g at 4°C. To pellet nuclei, the supernatant was taken from the low speed spin and sedimented for 10 minutes at 2000g at 4°C. Meanwhile, more nuclei were released by a second round of 25 strokes with pestle B, a short rest on ice, and 25 more strokes with pestle B. Debris was pelleted as above, and nuclei were pelleted as above in the same tube as the previous nuclei pellet. Nuclei yields were further increased with light sonication. Debris was resuspended in 300 μL total NPB and was sonicated in a Diagenode Bioruptor on Low power for 2 cycles of 30 seconds ON / 30 seconds OFF. Volume was brought to 1 mL total, and debris was removed and nuclei pelleted in the same tube as above. Nuclei were subjected to a buffer exchange and cleanup step to further remove any debris. The nuclei pellet was resuspended in 1 mL Tagmentation Reaction Buffer (TRB): 1 X Tagment DNA buffer (10 % Dimethylformamide, 10 mM Tris-HCl pH 7.5, 10 mM MgCl2), 0.33 X PBS, 0.01 % Digitonin, 0.1 % Tween-20. Debris was pelleted as above, and the supernatant was moved to a new tube and nuclei were pelleted as above. Nuclei were cleaned a final time by resuspending in TRB and pelleting. The supernatant was carefully removed to leave about 50 – 60 μL of supernatant without disturbing the nuclei pellet.  Nuclei were stained with DAPI and counted using a hemocytometer. Nuclei were tagmented by taking an aliquot of 50,000 nuclei, bringing the total reaction volume to 47.5 μL with TRB, and adding 2.5 μL TDE1 tagmentase. The tagmentation reaction was incubated for 30 minutes at 37°C with shaking. Immediately after the reaction concluded, reactions were cleaned up using the Zymo DNA Clean & Concentrator-5 kit.  Cleaned up tagmented DNA was added to a PCR reaction mix with 1.25 µM each Nextera i5 and i7 adapters and NEBNext High-fidelity 2X PCR Master Mix. Libraries were amplified with an initial extension and denaturation step of 5 minutes at 72°C and 30 seconds at 98°C followed by 12 cycles of 10 seconds at 98°C, 30 seconds at 63°C, and 1 minute at 72°C, with a final extension of 10 minutes at 72°C. Adapter contamination was initially removed with the Zymo DNA Clean & Concentrator-5 kit, and library size distributions were checked by running on a gel. Libraries were subjected to a final cleanup with 1.8 X AMPure XP beads.