title: GSM4491246: HF-3035_ChIP-free-chia-pet; Homo sapiens; OTHER
library_construction_protocol: 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 decrosslinked. The decrosslinked 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.
attributes.tag: GEO Accession
attributes.value: GSM4491246