title: GSM3143600: RNApolII in situ ChIA-PET, Wild type cultured NSC (TR3); Mus musculus; ChIA-PET
library_construction_protocol: For the in situ RNAPolII ChIA-PET, 10 million for formaldehyde crosslinked cells were suspended with 100  μl 0.55% SDS and incubated at RT for 10 min, 62°C for 10 min and 37°C for 10 min to permeabilize nuclei, which was followed by adding 25 μl 20% TritonX-100 with an incubation of 30 min at 37°C to quench SDS, and then 50 μl of blunt-end four-cutter AluI (cat# R0137L, NEB), 50 μl 10 × CutSmart buffer and 275 μl H2O incubating at 37°C overnight for in situ digestion. After pelleted and washed once with 1 ml 1× CutSmart buffer, the nuclei were suspended in 500 μl A tailing solution composed of 50 μl 10 × CutSmart buffer, 10 μl BSA (cat#B9000S, NEB), 10 μl 10 mM dATP (cat#N0440S, NEB), 10 μl Klenow (3'-5' exo-) (cat#M0202L, NEB), and 420 μl H2O and incubated at RT for 1 hr.  The in situ proximity ligation with biotinylated bridge linker was performed by adding 200 μl 5 × ligation buffer (cat#B6058S, NEB), 6 μl Bridge linker (200 ng/ul), 10 μl T4 DNA ligase (cat#M0202L, NEB) and 284 μl H2O and incubating at 16°C overnight. The nuclei with in situ proximity ligation were then subjected to sonication and chromatin immunoprecipitation with anti-RNApolII antibody (8WG16, cat# 920102, Biolegend, San Diego, CA), Tn5 tagmentation, biotin selection, PETs were amplified by PCR and sequenced.
attributes.tag: GEO Accession
attributes.value: GSM3143600