Instrument: Illumina HiSeq 4000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: 3C-libraries were prepared from homozygous E11.5 forelimb and hindlimb buds as described previously (Hagege et al. 2007). cHi-C experiments were performed as quadruplets (WT) or duplicates (mutants). Per biological replicate, 5-6 pairs of limb buds (ca. 3 ×106 cells) were micro dissected in PBS at room temperature (RT). A single cell suspension was obtained by incubating the tissue in 500 µl Trypsin-EDTA 0.05 % (Gibco) at 37°C for 10 minutes shaking at 900 RPM. The cells were resuspended and homogenised using a 0.40 µm cell strainer (Falcon) and diluted in 10% FCS/PBS. Cells were fixed by adding 650 µl 37% formaldehyde (Sigma-Aldrich) with a final concentration of 2% and mixed for 10 minutes at room temperature. Fixation was quenched using 1.425 M glycine (Merck) on ice and immediately centrifuged at 260 g for 8 minutes. Supernatant was removed, the pellet resuspended in lysis buffer (final concentration of 10 mM Tris pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 M EGTA, and 1x Complete protease inhibitors (Roche)) and incubated on ice for 10 minutes. Removal of lysis buffer was done by centrifugation at 400 g for 5 minutes at 4°C, followed by removal of supernatant, snap-freezing and storage at -80°C. On the next day, the pellet was resuspended in 520 µl 1x DpnII buffer (Thermo Fisher Scientific), and incubated with 7.4 µl 20% SDS shaking at 900 RPM at 37°C for 1 hour. Next, 75 µl 20% x-100 Triton was added and left shaking at 900 RPM at 37°C for 1 hour. A 15 µl aliquot was taken as a control for undigested chromatin (stored at -20°C). The chromatin was digested using 40 µl 10 U/µl DpnII (Thermo Fisher Scientific) shaking at 900 RPM at 37°C for 6 hours. 40 µl of DpnII was added and samples were incubated overnight shaking at 900 RPM at 37°C. On the third day, 20 µl DpnII was added to the samples shaking 5 more hours at 900 RPM at 37°C. DpnII restriction enzyme was inactivated at 65°C for 25 minutes. A 50 µl aliquot was taken to test digestion efficiency (stored at -20°C). Next, the digested chromatin was diluted and re-ligated in 5.1 ml H2O, 700 µl 10x ligation buffer (Fermentas), 5 µl 30 U/µl T4-ligase (Fermentas), incubated at 16°C for 4 hours and shake manually 3 times. The ligated samples were incubated further 30 minutes at RT. The chimeric chromatin products and test aliquots were de-crosslinked o.n. by adding 30 µl and 5 µl Proteinase K, respectively, and incubated at 65°C overnight. On the fourth day, 30 µl or 5 µl of 10 mg/ml RNase was added to the samples and aliquots, respectively, and incubated for 45 minutes at 37°C. Next, chromatin was precipitated by adding 1 volume phenol-chloroform to the samples and aliquots, vigorously shaking them, followed by centrifugation at 4000 RPM at RT for 15 minutes. The upper phase containing the chromatin was transferred to a new tube. To the aliquots, 100% ethanol was added and the samples were frozen for 30 minutes, centrifuged at 5000 RPM for 45 minutes at 4°C, washed with 70% ethanol and resuspended in 20 µl 10 mM Tris-HCl pH 7.5. To the samples, 7 ml H2O, 1 ml 3M NaAc pH 5.6 and 35 ml 100% ethanol was added. The samples were frozen at -20°C for 3 hours. The precipitated chromatin was isolated by centrifugation at 5000 RPM for 45 minutes at 4°C. The chromatin pellet was washed with 70% ethanol, and further centrifuged at 5000 RPM for 15 minutes at 4°C. Finally, the 3C-library chromatin pellet was dried at RT and resuspended in 10 mM Tris-HCl pH 7.5. To check the 3C-library, 600ng was loaded on a 1% gel together with the undigested and digested aliquots. The 3C-library was then sheared using a Covaris sonicator (duty cycle: 10%, intensity: 5, cycles per burst: 200, time: 6 cycles of 60 s each, set mode: frequency sweeping, temperature: 4 to 7 °C). Adaptors were added to the sheared DNA and amplified according to Agilent instructions for Illumina sequencing. The library was hybridized to the custom-designed sure-select beads and indexed for sequencing (50 to 100 bp paired-end) following Agilent instructions.