GSM9557552: PCHi-C_PA2_E11.5_Hoxa2PTE-42kb-del_2; Mus musculus; OTHER
Tissue from E8.5, E10.5 and E11.5 embryos was dissociated as described for RNA-seq. Then the dissociated cells were cross-linked with 2% paraformaldehyde for 10 min at room temperature (RT) and quenched with 125 mM glycine for 5 min at RT. Cells were spun down (1600 rpm, 10 minutes, 4 °C) and resuspended in cold 1× PBS. CNCCs from Wnt1::Cre;ROSARFP and Ezh2cKO embryos were filtered and collected by FACS. Cells were spun down (1600 rpm, 10 minutes, 4°C), supernatant removed, and cell pellets were flash-frozen and stored at -80°C. Promoter capture Hi-C was performed as described previously {Schoenfelder, 2018}  with adaptations to our system. The input material per PCHi-C experiment was 2.5 – 5 × 10^6 cells. Cells were incubated in 1mL ice-cold lysis buffer (10 mM Tris-HCl pH 8, 0.2% (vol/vol) NP-40, 10 mM NaCl,1× protease inhibitor cocktail (Roche)) for 30 min with periodical mixing, followed by centrifugation to pellet nuclei (760xg, 5 min., 4°C). Cell nuclei were resuspended in 358 µL ice-cold 1.25× NEBuffer 2, 11 µL of 10% SDS was added, and nuclei were incubated at 37°C, shaking at 950 rpm, for 1 hour. Then 75 µL of 10% Triton X-100 was added and cell nuclei incubated at 37°C, shaking at 950 rpm, for 1 hour. A 5 μL aliquot was taken as a control for undigested chromatin (stored at − 20 °C). For HindIII digestion 10 µL of HindIII enzyme (NEB, cat. no. R0104T) was added, incubated 6 hours at 37°C shaking at 950 rpm, followed by addition of another 10 µL of HindIII and overnight incubation at 37°C shaking at 950 rpm. A 5 μL aliquot was taken to test digestion efficiency (stored at − 20 °C). To fill in and biotinylate the restriction fragment overhangs 60 µL of biotinylation master mix (6 µL 10× NEBuffer 2, 2 µL H2O, 1.5 µL 10 mM dATP, 1.5 µL 10 mM dGTP, 1.5 µL 10 mM dTTP, 37.5 µL 0.4 mM biotin-14-dATP, and 10 µL 5U/μl Klenow (DNA polymerase I large fragment, NEB M0210L)) was added, incubated for 75 min at 37°C mixing regularly by pipetting, and then placed on ice. Next, 454 µL ligation master mix (100 µL 10× ligation buffer (NEB B0202), 10 µL 10 mg/mL BSA, 344 µL nuclease-free H2O) and 25 µL 1U/µL T4 DNA ligase (Invitrogen 15224-024) was added for overnight blunt end ligation in a water bath at 16°C. The next day ligated samples were incubated for a further 30 min at room temperature. The re-ligated chromatin products and test aliquots were de-cross-linked for at least 6 hours by adding 100 μL and 2.5 μL proteinase K (10 mg/mL), incubated at 65 °C. Afterwards, 12 μL or 0.5 μL of 10 mg/ml RNase A was added to the samples and test aliquots, respectively, and incubated for 60 min at 37 °C. Next, chromatin was precipitated by adding 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1) (PC) to the samples and test aliquots, vigorously shaking them, followed by centrifugation at maximum speed of a table top centrifuge (~ 13,000 rpm) at room temperature for 5 min. The upper phase containing the chromatin was transferred to a new 15 mL tube. To reduce loss of DNA 1 mL of TLE buffer (10 mM Tris HCl (pH 8.0), 0.1 mM EDTA) was added to tube containing PC and residual chromatin, tube was vigorously mixed, followed by centrifugation at maximum speed of a table top centrifuge (~ 13,000 rpm) at room temperature for 5 min. The upper phase containing the chromatin was transferred to the 15 mL tube. One tenth volume of 3 M sodium acetate was added to the chromatin sample mixed by vortexing, followed by the addition of 3 volumes 100% ethanol and vortexing to mix. The samples were frozen at − 80 °C for at least 2h. The precipitated chromatin was isolated by centrifugation at maximum speed for 45 min at 4 °C. The chromatin pellet was washed twice with 2mL freshly prepared 70% ethanol and centrifuged at maximum speed for 15 min at 4 °C in between washes. Finally, the chromatin pellet was dried at room temperature and resuspended in 100 μL TLE buffer. To check the Hi-C library DNA, 1:50 dilution of the sample, and the undigested and digested controls were loaded on a 1.5% agarose gel. The DNA concentration of the Hi-C library was determined using Qubit™ dsDNA HS assay kit.For PCHi-C 10 µg DNA (split into two 5 µg aliquots) were used for library preparation. The next steps were performed per 5 µg aliquot. The DNA was sonicated using Covaris S220 (peak incidence power: 175W; duty factor: 10%; cycles per burst: 200; time: 120s) in a volume of 130 µL in a Covaris microTUBE to obtain 100 – 1000 bp long DNA fragments. End repair, dATP-tailing and Adapter ligation was performed on beads. For this, 100 µL of Dynabeads™ MyOne™ Streptavidin T1 (Invitrogen) were washed twice with 400 µL Tween Buffer (TWB; 5 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween 20) and resuspended in 400 µL 2× binding buffer (2×BB; 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl). The volume of the sonicated DNA was increased to 400 µL with TLE. DNA was mixed with washed beads (800 µL total volume) and incubated at RT for 45 min rotating at 5 rpm. Beads were reclaimed on a magnetic separation stand, supernatant was removed, followed by a wash with 400 µL 1×BB, then with 100 µL 1×ligation buffer, and resuspended in 50 µL 1× ligation buffer. For end repair each aliquot was mixed on ice with 50 µL 2.5 mM dNTP mix (12.5 μL of 10 mM of each dNTP), 18 μL of  T4 DNA Polymerase (NEB M0203), 18 μL of T4 PNK (NEB M0201), 3.7 μL of DNA polymerase I large fragment (Klenow; NEB M0210), and 360.1 μL of H2O, and then incubated at 20°C for 60 min. Beads were reclaimed on a magnetic separation stand, supernatant was removed, followed by two washes with 500 µL 1×TWB, one wash with 500 µL 1×BB, one wash with 500 µL TLE and resuspended in 415 µL TLE. For dATP-tailing each aliquot was mixed with 50 µL 10× NEBuffer 2, 5 µL 10 mM dATG and 20 µL 5 U/µL Klenow exo-minus, and then incubated at 37°C for 30 min. Beads were reclaimed on a magnetic separation stand, supernatant was removed, followed by two washes with 500 µL 1×TWB, one wash with 500 µL 1×BB, one wash with 200 µL 1× ligation buffer and resuspended in 100 µL 1× ligation buffer. For PCHi-C adaptor ligation 4 µL 15 µM pre-annealed PE adapters (described in Schoenfelder et al., 2018) and 4 µL NEB T4 Ligase 400 U/mL (NEB, M0202) were added to each aliquot and incubated 2 h at room temperature. For Hi-C 10 µL 15 µM TruSeq DNA Sgl index adapters (Illumina, Cat. No. 20016329) and 4 µL NEB T4 Ligase 400 U/mL (NEB, M0202) were added to each aliquot and incubated 2 h at room temperature. Beads were reclaimed on a magnetic separation stand, supernatant was removed, followed by two washes with 500 µL 1×TWB, one wash with 500 µL 1× BB, one wash with 200 µL 1×BB, one wash with 100 µL 1× NEBuffer 2, one wash with 50 µL 1× NEBuffer 2 and resuspended in 50 µL 1× NEBuffer 2. At this point all aliquots per PCHi-C sample were pooled. For on-bead PCHi-C library amplification a reaction master mix was prepared, reaction volume per PCR tube was 25 µL (PCHi-C: 5 µL 5× Herculase II reaction buffer (Agilent), 0.25 µL 100 mM dNTPs, 2 µL PE PCR primer 1.0 {Schoenfelder, 2018}, 2 µL PE PCR primer 2.0 {Schoenfelder, 2018}, 0.5 µL Polymerase (Herculase, Agilent), 2.5 µL Hi-C DNA on beads, 12.75 µL nuclease-free H2O; Hi-C: 12.5 µL KAPA HiFi HotStart ReadyMix (Roche), 2.5 µL Illumina primer mix (contained in KAPA HiFi HotStart Library Amplification Kit), 2.5 µL Hi-C DNA on beads, 7.5 µL nuclease-free H2O) and PCR was run under the following conditions: 30s at 98 °C; 6-7 cycles of: 10 s at 98 °C, 30 s at 65 °C, 30 s at 72 °C; 7 min at 72 °C. Post-amplification PCR reactions per sample were pooled and DNA was cleaned up using AMPure XP beads and eluted in nuclease-free H2O. Obtained DNA was used directly for Hi-C sequencing or for promoter-capture as described in Schoenfelder et al., 2018. Sequencing was performed on an Illumina NextSeq 500 (75bp read length, paired-end).