GSM3261973: CHiC-Inv1DelCTCF-Distal-E115; Mus musculus; OTHER
Preparation and fixation of limb tissue and ESCs was performed according to the instructions of the Hi-C protocol. Fixed cell pellets were resuspended in cHi-C 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. Lysis buffer was removed by centrifugation at 400 g for 5 minutes at 4°C, succeeding to removal of supernatant, snap-freezing and storage at -80°C. The following day, the thawed pellet was resuspended in 360 µl H2O, 60 µl 10x DpnII buffer and 15 µl 10% SDS. After 1 h of shaking with 900 rpm at 37°C 150 µl of 10% Triton-X-100 were added and the mixture incubated for another hour with the same conditions. 15 µl were taken as a control for undigested chromatin (stored at -20°C). Then, digestion of chromatin was initiated by adding 40 µl DpnII (10 U/µl, NEB R0543S) and incubated for 4 h at 37°C and shaking at 900 RPM. Chromatin was further digested by adding 30 µl DpnII and incubation of the sample overnight. The following day, another 30 µl DpnII were added and the samples shaken for 4 more hours. Inactivation of the enzyme was done by incubation at 65°C for 25 minutes. A 50 µl aliquot was taken to test digestion efficiency (stored at -20°C). Then, the library sample was transferred to a 50 ml falcon tube, diluted in 5.1 ml of water and mixed with 700 µl 10x T4-DNA-ligase buffer and 5 µl T4-DNA-ligase (30 U/µl, Thermo Fisher Scientific EL0013). After 4 hours of incubation in a 16°C water bath with manually shaking (once per hour) the sample was incubated for 30 min at RT. Through addition of 30 µl of Proteinase K, the samples were reverse-crosslinked overnight at 65°C. On the fourth day, RNA in the samples was digested by subjecting 30 µl of 10 mg/ml RNase, incubating 45 minutes at 37°C. Subsequent to addition of 7 ml phenol-chloroform, samples were centrifuged 15 min at 2440 g and RT. The water phase was transferred into a new tube and mixed with 7 ml H2O, 1 ml of 3 M sodium acetate (pH 5.6) and 35 ml of 100% ethanol. For precipitation of DNA, samples were kept overnight at -20°C and spun down with 5250 g for 45 min at 4°C. After a wash with 10 ml of 70% ethanol and 15 min of centrifugation (5250 g, 4°C), the pellet was air-dried and dissolved in 150 µl 10 mM Tris-HCl (pH 7.5). In order to check the 3C-library, 600 ng of chimeric DNA was loaded on a 1% gel together with the undigested and digested aliquots. Next, the 3C-library was sheared using a Covaris sonicator with the conditions as for HiC (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). After adaptors were added to the sheared DNA,  amplification was performed according to Agilent instructions for Illumina sequencing. The library was hybridized to the custom-designed sure-select probes and indexed for sequencing (50 to 100 bp paired-end) as instructed by Agilent protocols.
GEO Accession
GSM3261973