GSM3261967: HiC_WT_E115-LB-Rep3; Mus musculus; Hi-C
The HiC protocol was adapted from Rao et al., 2014. 3C-libraries were prepared from E11.5 limb buds as described previously (Hagege et al. 2007). HiC was performed as technical triplicates from limb buds of 8 embryos in total. After pooling the dissected tissue, it was turned into a single-cell suspension by digestion with Trypsin-EDTA 0.05% (Gibco) for 10 min at 37°C, disrupting the tissue by pipetting every 2 minutes. The cells were diluted in 10% FCS/PBS and homogenized using a 40 µm cell strainer (Falcon). Cells were pelleted by centrifugation (260 g, 5 minutes), resuspended in 10% FCS/PBS and fixed by adding 37% formaldehyde (Sigma-Aldrich) with a final concentration of 2% and put on a roller machine for 10 minutes at RT. By adding 1.425 M glycine (Merck) on ice,  fixation was quenched and the samples were immediately centrifuged at 260 g for 8 minutes. Supernatant was aspirated, the pellet resuspended in HiC lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1.15% Triton-X-100 and 1x Complete protease inhibitors (Roche)), incubated for 10 minutes on ice. Nuclei were pelleted, snap-frozen in liquid nitrogen and stored at -80°C. Frozen pellets were washed with 500 µl  DpnII buffer (NEB) and centrifuged for 5 minutes at 4°C. Then, the pellet was resuspended in 50 µl of 0.5% SDS, incubated for 10 minutes at 62°C, followed by addition of 145 µl of water, 25 µl of 10% Triton-X-100 (Sigma, 93443) and incubation at 37°C for 15 minutes. After taking a 15 µl aliquot as a control for undigested chromatin, 25 µl of 10x DpnII buffer and 10 µl of DpnII (100 U/µl; NEB) were added to the samples and chromatin was digested overnight at 37°C, rotating in an oven. The DpnII enzyme was inactived by incubation at 65°C for 25 minutes. An aliquot of 15 µl was taken to test digestion efficiency. Subsequent to pelleting the samples (5000 g, 5 minutes) and resuspending them in 200 µl of 1.2x NEBuffer 2, 50 µl of fill-in-mastermix (0.3 mM biotin-14-dATP (Life Technologies 19524-016), 0.3 mM dCTP, 0.3 mM dGTP, 0.3 mM dTTP, 0.8 U/µl DNA Polymerase I Large (Klenow), Fragment (NEB, M0210)) were added and samples were incubated at 37°C for 90 minutes with constant rotation in an oven. 900 µl of ligation-mastermix (1.33x NEB T4 DNA ligase buffer (NEB, B0202), 1% Triton-X-100, 0.13 mg/ml BSA, 2.2 U/µl T4 DNA ligase (NEB, M0202)) were added, mixed by inverting the samples and incubated at 18°C overnight with slow rotation in an oven. The next day, 25 µl were taken as ligation control and samples were spun down at 5000 g for 5 minutes. After the pellets were resuspended in 600 µl of Tris-HCl (10 mM, pH 7.5), de-crosslinking was performed by addition of 25 µl of proteinase K and 60 µl of 10% SDS, and incubation at 55°C for 30 minutes, succeeded by addition of NaCl and incubation at 68°C for 2 hours. DNA was precipitated by mixing the samples with 750 µl of pure ethanol and 75 µl of 3M sodium acetate (pH 5.2), followed by incubation at -80°C for 20 minutes. The precipitated DNA was pelleted by centrifugation at 16000 g for 15 minutes at 2°C. Samples were washed twice with 800 µl of 70% ethanol, then resuspended in 130 µl Tris-HCl (pH 8) and incubated at 37°C for 15 minutes to fully dissolve the DNA. Succeeding to shearing the DNA to a size of 300-500 bp with 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), the samples were transferred to low-bind tubes and diluted in 100 µl of water. Per sample, 150 µl of Dynabeads MyOne Streptavidin T1 beads(Life technologies, 65602) were  washed in  Tween Washing Buffer (TWB: 5 mM Tris-HCl (pH 7.5), 1 M NaCl, 0.5 mM EDTA, 0.5% Tween-20), resuspended in 300 µl 2X Binding Buffer (BB: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2M NaC), mixed with the sheared DNA and incubated at RT for 15 min with rotation to bind biotinylated DNA. The beads were washed twice with 600 µl TWB, (for all washing steps with TWB, the tubes are heated to 55° for 2 minutes) and afterwards washed once with T4 DNA ligase buffer (NEB, B0202). In order to repair the sheared ends of DNA and to remove biotin from unligated ends, the beads were resuspended in 100 µl of mastermix (0.88x NEB T4 DNA ligase buffer with 10 mM ATP, 0.5 mM dNTP mix, 0.5 U/µl PNK (NEB, M0201), 0.07 U/µl T4 DNA polymerase (NEB, M0203), 0.05 U/µl DNA polymerase I, Large (Klenow) Fragment (NEB M0210)) and incubated for 30 minutes at RT.  Beads were washed twice with 600 µl of TWB and transferred to a new low-bind tube. Subsequent to washing the beads once with 200 µl NEBuffer 2, they were resuspended in 100 µl dATP attachment master mix (0.9X NEBuffer 2, 0.5 mM ATP, 0.25 NEB Klenow exo minus (NEB, M0212) and incubated for 30 minutes at 37°C. Again, the beads were washed twice with TWB,  once with 100 µl of NEB Quick ligation buffer (NEB B6058) and transferred to a new tube. After resuspension of the beads in 50 µl of NEB Quick ligation buffer, 2 µl of NEB DNA Quick ligase (NEB M2200) and 3 µl of universal adapter from NEBNext® Ultra™ DNA Library Prep Kit for Illumina and incubated at RT for 15 minutes. The suspension was mixed with 3 µl of User enzyme and incubated at 37°C for 15 minutes. Thereafter, beads were washed twice with TWB, washed once with 100 µl 10 mM Tris-HCl (pH 7.5) and transferred to a new low-bind tube, and finally resuspended in 50 µl 10 mM Tris-HCl (pH 7.5). Next, the HiC library was directly amplified from the T1 beads according to Illumina protocols (Illumina, 2007). After amplification was complete, the volume of the library was adjusted to 250 µl with 10 mM Tris-HCl (pH 7.5). Following to addition of 137.5 µl of AMPure XP beads, the reaction mix was incubated 5 minutes at RT. Beads were reclaimed via a magnet and the supernatant was again subjected to 37.5 µl of AMPure XP beads with 5 minutes incubation at RT. Beads were captured via a magnet and washed twice with 700 µl of 70% ethanol. After air-drying the beads, the bound DNA was eluted by addition of 25 µl of Tris (10 mM, pH 7.5) and incubation for 5 minutes at RT. Once again, the beads were separated with a magnet and the supernatant transferred to a new tube. The libraries were sequenced on an Illumina HiSeq4000 with ca. 200 million reads (75 bp, paired-end) per sample.
GEO Accession
GSM3261967