Worm_embryo_extraction_vRG2. Fixed embryos were suspended in 8mL of ChIP buffer (50 mM Hepes-KOH pH 7.6, 140 NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 % NP-40) and sonicated on ice with a Branson sonifier microtip (25 % power setting for 2 min; 30 % power setting for 2 min; 35 % power setting for 10 min; 40 % power setting for 2 min; 45 % power setting for 2 min). Debris were pelleted at 10 000 g for 20 min and the supernatant was made 10 % in glycerol. The extract was snap frozen in liquid nitrogen aliquots containing 3 mg of protein and stored at ? 80 °C. For a detailed protocol see http://www.modencode.org/. Worm_chromatin_immunoprecipitation_vRG2. For each ChIP reaction, 3 mg of protein extract was diluted with ChIP buffer (50 mM Hepes KOH pH 7.6, 140 NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 % NP-40) to 900 ?l. Then 35 ?l 30 % sarcosyl (1 % final) and 20 ?l 5 % Na-deoxycholate (0.1 % final) were added. 50 ?l of the diluted extract were removed (input sample), mixed with Elution buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 250 mM NaCl, 1 % SDS) and processed as described for ChIP samples. 100 ?l antibody/bead suspension (5?g antibody pre-bound to 50 ?l Dynabeads, suspended in ChIP buffer) was added and the mixture and rotated at least 1.5 hr to over night at 4 °C. Beads were washed with 2 × 1 ml FA Buffer (50 mM Hepes-KOH pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % Na-deoxycholate) for 5 min each; with 1 ml FA-1000 buffer (50 mM Hepes-KOH pH 7.6, 1 M NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % Na-deoxycholate) for 10 min; with 1 ml FA-500 buffer (50 mM Hepes-KOH pH 7.6, 500 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % Na-deoxycholate) for 10 min; with 1 ml TEL buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1 mM EDTA, 1% NP-40, 1% Na-deoxycholate) for 10 min; and briefly with 1 ml TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA). Immunocomplexes were eluted with 50 ?l Elution buffer for 15 min at 67 °C. Eluates were incubated over night at 65 °C to reverse cross-links, then treated with Proteinase K (0.44 mg/ml) at 37 °C for 2 h. Nucleic acids were recovered by phenol/chloroform extraction and ethanol precipitation. After digestion with RNAse A (0.33 mg/ml) for 2h at 37 °C, DNA was purified using the Qiagen PCR purification kit. For a detailed protocol see http://www.modencode.org/. DNA for Library Prep is incubated with an End Repair Enzyme mix (NEB Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends. It is then purified and incubated with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends (a single A-overhang for more efficient and directed ligation of the adaptors). After a second purification, the DNA fragments is ligated with single-end ?Homebrew? adaptors which contain an index sequence within (Corbin Jones? lab). After ligation, the samples are purified TWICE using SPRI beads, allowing for a size selection step getting rid of excess adaptors. Samples are then amplified by PCR with single end primers. Depending on the original fragmentation of the DNA, samples can either be purified by SPRI or by separation and purification from an agarose gel. Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.