GSM5399312: MED4_degron_0h_PCHi-C_20k_genes_Rep1; Mus musculus; Hi-C
Five million cells were harvested and formaldehyde-crosslinked as for ChIP-seq. Crosslinked cells were lysed for 15 min on ice in 10 mM Tris-HCl pH 8.0, 10 mM NaCl, and 0.2% NP-40 supplemented with cOmplete protease inhibitor. Nuclei were isolated by centrifugation and by removing the supernatant. Nuclei were gently resuspended in 50 μL of 0.5% SDS and incubated at 62°C for 5-10 min. SDS was quenched by adding 170 μl of 1.5% Triton X-100 and incubating at 37°C for 15 min. 25 μl of 10×NEBuffer2 and 100 U of MboI restriction enzyme were added and the sample was digested overnight on a thermo-mixer. MboI was inactivated by heating to 62°C for 20 min. After the sample was cooled down to room temperature, a fill-in master mix (37.5 μl of 0.4 mM biotin-14-dATP, 1.5 μl of 10 mM dCTP, 1.5 μl of 10 mM dGTP, 1.5 μl of 10 mM dCTP, 8 μl of Klenow Fragment) was added to the sample and incubated at 37°C for 1 hour. Proximity ligation was carried out by adding a ligation master mix (663 μl of H2O, 120 μl of 10X NEB T4 DNA ligase buffer, 100 μl of 10% Triton X-100, 12 μl of 10 mg/ml BSA, 5 μl of T4 DNA ligase) and incubating for 4 hours at room temperature. The sample was treated with Proteinase K before DNA purification with ethanol precipitation. Purified DNA was sheared on a Qsonica Q800R2 sonicator with 20% amplitude for 9 cycles at 10 s each with 30 s between cycles at 4°C. Size-selection of the sheared DNA was performed with KAPA pure beads to enrich fragments in the range of 300-500 bp. Size-selected biotinylated DNA was pulled down with Dynabeads MyOne Streptavidin T1 beads. After on-bead end repair and A-tailing, SureSelect Adaptor was added to the ends of DNA following Agilent's protocol. The adaptor-ligated DNA was amplified directly off the beads with 6-8 PCR cycles. PCR products were purified and concentrated to 221 ng/μl using a vacuum concentrator. For capture, two 120-nt biotinylated RNA probes were designed to anneal specificity to the promoter region of one gene. In total, ~40,000 RNA probes targeting ~20,000 mouse genes were synthesized at Agilent. 3.4 μl (750 ng) of the concentrated PCR products were hybridized to the RNA probes following Agilent's protocol. The hybridized library was captured by Dynabeads MyOne Streptavidin T1 beads and amplified off the beads with Agilent's indexing primers. Libraries were constructed using Agilent's SureSelect library prep kit