GSM3553630: HF-3177_chia-pet; Homo sapiens; ChIA-PET
10 million cells were dual-crosslinked with 1.5mM EGS (21565, Thermo Fisher) for 45 min followed by 1% formaldehyde (F8775, Sigma) for 20 min at room temperature and then quenched with 0.125 M Glycine (G8898, Sigma) for 10 min. The crosslinked cells were washed with PBS twice and lysed in 100 ul 0.55% SDS with incubation at room temperature, 62°C and 37°C sequentially for 10 min each, which was followed by 37°C for 30 min with addition of 25 ul 25% Triton-X 100 to quench SDS and 37°C overnight with addition of 50 ul AluI (R0137L, NEB), 50 μl 10 × CutSmart buffer and 275 μl H2O to fragmentize the chromatin. The pelleted digested nuclei were resuspended in 500 ul A-tailing solution containing 50 μl 10 × CutSmart buffer, 10 μl BSA (B9000S, NEB), 10 μl 10 mM dATP (N0440S, NEB), 10 μl Klenow (3'- 5' exo-) (M0202L, NEB), and 420 μl H2O  with 1 hr incubation at room temperature and then subjected to proximity ligation by adding 200 μl 5 × ligation buffer (B6058S, NEB), 6 μl biotinylated bridge linker (200 ng/ul), 10 μl T4 DNA ligase (M0202L, NEB) and 284 μl H2O and incubating at 16°C overnight. The ligated chromatins were then sheared by sonication and immunoprecipitated with anti-RNApolII antibody (920102, Biolegend). The ChIPped DNA were tagmented using the Nextera DNA Sample Preparation Kit (Illumina). The linker ligated ChIA-PET constructs were selected using streptavidin magnetic beads. The magnetic beads were blocked with sheared gDNA prior to the selection. The streptavidin selected constructs were amplified with PCR and purified.