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This ChIP protocol for embryonic tissues is adapted from (Visel et al., 2009a) for tissue collection and crosslinked chromatin preparation, and on (Kim et al., 2007) and (Vokes et al., 2008) for subsequent steps, and has been modified and optimized by Marco Osterwalder and Javier Lopez-Rios. Embryos were collected in ice-cold PBS, and dissected tissues (forelimbs/hindlimbs or hearts) from somites-matched embryos of the same genotype were pooled and collected in ice-cold PBS into 2ml Eppendorf tubes. Dissected tissues were washed 2 times with ice-cold DPBS containing Ca2+ and Mg2+ (Gibco) and transferred to a 2ml glass douncer (Tissue Grind Tube Size 2ml, Kimble-Chase) on ice. Tissue disaggregation was achieved by applying 25 strokes with pestle A (Tissue Grind Pestle LC 2ml, Kimble-Chase) followed by 25 strokes with pestle B (Tissue Grind Pestle SC 2ml, Kimble-Chase). The solution containing nuclei was transferred back to a 2ml Eppendorf tube, and the douncer was rinsed with 0.3ml cold DPBS w/Ca2+Mg2+ that was also added to the sample. After centrifugation at 3000 rpm for 3min (4°C), the supernatant was discarded and nuclei were resuspended in 1.5ml RT DPBS w/Ca2+Mg2+ containing 150µl 11x crosslinking buffer (0.1M NaCl, 1mM EDTA, 0.5mM EGTA, 50mM Hepes pH 8.0; add 11% formaldehyde just before use). The mixture was incubated for 5min at RT with gentle horizontal shaking. To stop crosslinking, 75µl of 2.5M Glycine were added, followed by another incubation of 5min at RT with horizontal shaking. The samples were then

centrifuged at 3000 rpm for 3min (4°C), supernatants removed, and pellets resuspended in 1.5ml ice-cold DPBS w/Ca2+Mg2+. After re-centrifugation at 3000 rpm for 3min (4°C), the supernatants were discarded and the nuclear pellets snap-frozen in liquid N2 and stored at -80°C. In order to use sufficient material for the ChIP, crosslinked nuclear pellets from different embryo collections can be pooled in one single sample. Generally, 3 samples containing crosslinked nuclei from similar amounts of dissected embryos were processed for ChIP in parallel in one experiment.

Before performing the ChIP, antibodies coupled to magnetic beads were prepared. For one ChIP sample, 20µl of Dynabeads® Protein G were used and transferred to a 2ml Eppendorf tube containing 1ml freshly prepared ice-cold BSA (5mg/ml in DPBS w/Ca2+Mg2+). Beads were washed 6 times with 1ml cold BSA/PBS using a magnetic rack, resuspended in a volume corresponding to 2.5x the original volume of beads (50µl per sample) and transferred to a 2ml screw cap microtube (Starstedt). 2µg of anti-FLAG M2 antibody were used for each ChIP sample and incubated overnight on a rotating wheel at 4°C.

The following day, the Dynabeads-antibodies complexes were transferred back to a 2ml Eppendorf tube and washed 6 times with 1ml cold BSA/PBS before being resuspended in the original volume of suspended beads (20µl per sample) and kept on ice. In the meantime, snap-frozen crosslinked nuclear pellets were thawed on ice and resuspended in 6ml cold Lysis buffer (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8.0, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x Complete Mini protease inhibitor cocktail (Roche)). If necessary, several crosslinked nuclear pellets were pooled and resuspended in 6ml of ice-cold Lysis buffer (see comment above). Lysates were incubated for 10min at 4°C with gentle rocking, followed by 10min centrifugation at 2500 rpm (4°C). Supernatants were discarded and the pellets resuspended in 2ml of RT Protein Extraction Buffer (0.2M NaCl, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 10mM Tris-HCl pH 8.0, 1x Complete Mini protease inhibitor cocktail) to which 3ml of Protein Extraction Buffer were added. The samples were incubated for 10min at RT on a rocking platform and the nuclei were pelleted by centrifuging 10min at 2500 rpm at 4°C. After discarding the supernatant, the nuclei were resuspended in 1ml ice-cold Chromatin Extraction Buffer (1mM EDTA pH 8.0,

0.5mM EGTA pH 8.0, 10mM Tris-HCl pH 8.0, 0.1% Na-Deoxycholate, 0.5% N- lauroylsarcosine, 3x Complete Mini protease inhibitor cocktail). Samples were then transferred to Covaris TC 12x12 tubes and sonicated to shear the crosslinked chromatin using the S220 Covaris Ultra-sonicator. Each sample was sonicated for 15min using the 5% duty cycle, 140 watts Peak Incident Power and 200 Cycles per Burst. The sheared chromatin was then transferred back to 1.5ml Eppendorf tubes, and insoluble debris were pelleted by centrifuging for 10min at 13’000 rpm (4°C). The supernatants were moved to new 2ml screw cap microtubes, and the volume was topped up to 1.060ml with Chromatin Extraction Buffer. At this stage, 2x 30µl aliquots were taken and separated to new Eppendorf tubes: one for input control and the other to assess the quality of the sonicated chromatin on an agarose gel. Both aliquots were kept overnight at 4°C to be treated identical to the ChIP sample. Then, 300µl of ChIP cocktail Mix (130µl Triton X-100 10%, 3µl Na-Deoxycholate 10%, 26µl Complete protease inhibitor solution (from a 50x tablet dissolved in 1ml mQ H2O), 131µl TE buffer (100mM Tris HCl pH 7.4, 10mM EDTA pH 8.0) and 10µl mQ H2O were added to each 1ml ChIP sample to adjust to RIPA buffer conditions (see below). For each ChIP sample, 20µl of the freshly washed Dynabead-antibody complexes were added and incubated overnight on a rotating wheel (4°C).

The next day, the dynabeads were transferred to 1.5ml Eppendorf tubes containing 1ml of freshly prepared cold RIPA buffer (50mM Hepes pH 8.0, 1mM EDTA pH 8.0, 1% NP-40, 0.7% Na-Deoxycholate, 0.5M LiCl, 1x Complete Mini protease inhibitor cocktail), and subsequently washed 6 times with 1ml cold RIPA buffer on a magnetic rack. The beads were rinsed once with 1ml TE-plus (100mM Tris HCl pH 8.0, 10mM EDTA pH 8.0, 50mM NaCl, 1x Complete Mini protease inhibitor cocktail) and centrifuged for 3min at 1000 rpm (4°C). All residual liquid was removed from the beads using the magnetic rack, and the beads were resuspended in 100µl freshly prepared Elution Buffer (1mM EDTA, 1% SDS, 10mM Tris-HCl pH 8.0). Elution of the crosslinked protein-DNA complexes from the Dynabead-antibody complexes was performed at 65°C for 15min on a heat block shaking at 1300 rpm. Beads were then centrifuged for 1min at 13’000 rpm and retained in the tube using a magnetic rack, while 100µl of supernatant containing the protein-DNA complexes were transferred to a PCR microtube. In parallel, the 30µl input aliquot and the 30µl chromatin aliquot

(kept on ice) were also transferred to PCR microtubes containing 120µl of Elution Buffer. Reverse crosslinking was performed for all three samples by incubating them overnight at 65°C (≥12hrs).

The next day, samples were transferred back to 1.5ml Eppendorf tubes and treated with RNase A (from bovine pancreas, Sigma) in TE (100mM Tris HCl pH 8.0, 10mM EDTA pH 8.0) at a final concentration of 0.2µg/µl. After 1hr incubation at 37°C, Proteinase K was added to a final concentration of 0.2µg/µl and proteins were digested for 2hrs at 55°C. Finally, DNA was purified from all three samples using the QIAquick Gel Extraction Kit (Qiagen). Elution was achieved in two steps using 30µl Buffer EB each, and the eluted ChIP and input samples were aliqoted and stored at - 20°C before use for qPCR. To assess the quality of the sonication, half of the purified chromatin control samples were loaded on an agarose gel to estimate the range of DNA fragments sizes (between 150bp to ~1.5kb with an maximal intensity at ~300bp, data not shown).