Mus musculus
Protocols: For 4C libraries we collected the following embryonic mouse tissues: - C57Bl/6 wild-type forelimbs (dissected into anterior, middle and posterior sections) and whole hindlimbs of E11.5 mouse embryos - INV(6-C2)/del(-90kb-C2) whole forelimbs and hindlimbs. For this purpose heterozygous INV(6-C2) mice were crossed with heterozygous del(-90kb-ShhSB2) mice. Compound heterozygous embryos could phenotypically easily be distinguished from all other phenotypes due to their small size and truncated limbs. All tissues were collected into PBS pH=7.4. Single cell suspensions were obtained by incubation in 1% trypsin at 37° C for 1 minute and rigorous pipetting with a blue P1000 tip. The cells were then immediately fixed with 2% formaldehyde in 10% FCS/PBS at room temperature for 10 minutes. Glycine was added to a final concentration of 0.1M and the samples were moved to ice to quench the cross‐linking reaction. The cells were centrifuged at 8000 rpm for 8 minutes at 4°C, and the supernatant removed. Cells were lysed in 1ml cold lysis buffer (50mM Hepes pH=8, 150mM NaCl, 5mM EDTA, 0.5% NP‐40 1.2% Triton X‐100) for 10 minutes on ice. Successful lysis was verified by Methyl Green‐Pyronin staining. Cell lysate was centrifuged at 8000 rpm at 4°C, the supernatant removed, and the pellet was frozen in liquid N2 until sufficient material was available. Once sufficient tissue had been collected, cells were resuspended in 62.5 µl 10X NlaIII restriction buffer, placed at 37ºC and 15 µl 10% SDS was added. Cells were incubated for 1h at 37ºC, while shaking at 900rpm to remove non-crosslinked proteins. Subsequently, SDS was sequestered, by adding 150µl 10% Triton X-100 and incubated at 37ºC while shaking at 900 RPM. The sample was then digested by adding 400U (40 µl) NlaIII and incubation at 37ºC. After 4h and 8h another 400U of enzyme was added (total: 1200U NlaIII), adjusting the volume and buffer concentration as necessary, and the restriction digest was continued overnight , during the two latter digests. The next day we verified digestion efficiency by de-crosslinking and proteinaseK-treating samples taken before and after digest and running on an 0.6% agarose gel. If digest was not sufficient further enzyme was added for another 4h. Following sufficient digestion, enzyme was inactivated by incubating 20 minutes at 65°C and samples were transferred to a 50ml falcon tube. Samples were ligated in a 7ml volume using 700µl 10X ligation buffer (0.3M Tris‐HCl pH=7.8, 0.1M MgCl2, 1.54% DTT, 0.5% ATP) and 50U T4 DNA ligase and incubated at 16°C overnight. We determined ligation efficiency by de-crosslinking and proteinaseK-treating 100µl of ligation reaction and running on an 0.6% agarose gel. If further ligation was necessary, we added fresh ATP and continued ligation for 4-6h. Once ligation was complete we de-crosslinked the sample overnight at 65ºC in the presence of 30µl Proteinase K (10mg/ml). The next morning, we added 30µl RNAse A (10mg/ml) and incubated the sample for 45 min at 37ºC. We then extracted DNA by adding 7ml phenol-chloroform and precipitating DNA using 7ml water, 1.5 ml 2M NaAcetate pH 5.6, 7 µl 20mg/ml glycogen and 35 ml 100% ethanol. The resulting pellet was dissolved in 150µl Tris pH 7.5 at 37ºC. The obtained 3C library was then subjected to a second round of overnight digestion using 60U DpnII in a 500µl volume. The next day we verified digestion efficiency and subsequently inactivated the enzyme by incubating the sample at 65ºC for 25 minutes. The sample was then subjected to phenol-chloroform extraction, resuspended in 100µl water and ligated overnight with 100U T4 DNA ligase in a final volume of 14.3ml. The next day the DNA was first phenol-chloroform extracted and then purified using the QIAquick PCR purification kit, using 3 columns per sample, eluting with 50µl 10mM Tris per column. 4C primers for the region of interest were selected from the Tanay lab 4C primer database {vandeWerken:2012gv}, further tested for off-targets using BiSearch {Aranyi:2006ip} and only primers with <50 hits were kept. For quality assessment of primers we tested them on 25, 50, 100 and 200ng of 4C template and kept only those with reproducible banding pattern at all concentrations. The final list of 4C primers is included in the Primer Table. Using these primers, we set up PCR reactions for sequencing with primers containing barcodes and the Solexa sequencing adapter at their 5' end. For PCRs we used Expand Long Template system from Roche, using either 100 or 200ng of the 4C library as template and setting up 16 PCR reactions in parallel for each primer. PCR products were purified using Roche HighPure PCR product purification kit, adding an additional 1 min spin after the last wash step to remove all residual ethanol. The samples were eluted in 50μl 10 mM Tris pH=8.0, pooled and 1 µg each of approximately 40 PCR products were pooled to achieve a balanced base composition at the first 4 bases.