Nuclei were purified from frozen samples as described (Haenni et al. 2012). Briefly, samples were thawed on ice, and then mixed with 150 ml of 2X nuclei purification buffer (20mM Hepes pH 7.6, 20mM KCl, 3mM MgCl2, 2mM EGTA, 0.5 M Sucrose, 0.05% Triton, 1mM DTT, 0.05M NaF, 40mM b-glycerophosphate, 2mM Na3VO4). The samples were then transferred to a Wheaton stainless-steel homogenizer and were homogeneized with 3 plunger strokes. The resulting samples were centrifuged at 200 g for 1 min to remove worm debris. The supernatant was transferred to a fresh tube, and the remaining pellet was resuspended in 150 ml of 2X nuclei purification buffer and homogenized as described above. The process was repeated until no visible pieces of worm remained (approximately 5 times). The pooled supernatants were centrifuged at 200 g for 1 min to remove worm debris. Finally, the nuclei were pelleted at 1000 g for 10 min at 4°C. The purified nuclei were immediately used for the ATAC-seq protocol. Briefly, the nuclei were resuspended in 47.5 ml of Nextera Tagmentation buffer (Nextera DNA Sample Preparation Kit) and incubated with 2.5 ml of the Tn5 transposase at 37°C for 30 min. Resulting DNA fragments were purified using a miniElute column (Qiagen) and amplified by NEBNext High-Fidelity PCR Master Mix in a total volume of 50 ml. The thermocycling protocol for this reaction was 72°C for 5 min, 98°C for 30 s and 5 cycles of 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. Every sample shared the Adapter1 primer, and had a unique barcoded Adapter2 primer (Supplemental Table 7). To ensure over-amplification did not occur, after the initial 5 cycles, the number of remaining cycles required was estimated for each sample using qPCR (BioRad CFX96 Real-Time System). To do so, a similar reaction to the above was set up (NEBNext, Adapter1 and a single Adapter2), with the addition of SybrGreen, and using 5 ml of the previous PCR as template. The final volume was 15 ml, and the thermocyling was as above except that the initial incubation step was 98°C for 30 seconds, and 40 cycles were performed. The number of additional cycles was determined to be the number it took for the qPCR to reach one-third maximal fluorescence. The original PCR was then resumed and each sample cycled as necessary. Following amplification, the samples were purified using QIAquick columns (Qiagen), and library quality was verified on an Agilent bioanalyzer. Sequencing was performed using 101 bp paired-end sequencing on an Illumina Hi-seq 2000.