title: GSM3476504: Pachynema replicate 3; Mus musculus; Hi-C
library_construction_protocol: 600,000-800,000 cells were cross-linked with 2% formaldehyde for 10 min at room temperature, then the reaction was quenched using 200 mM glycine for 5 min at room temperature, then 15 minutes on ice, then samples were frozen in liquid nitrogen. Nuclei were isolated and directly applied for digestion using the 4-base cutter restriction enzyme MboI (NEB) at 37°C overnight. The single strand overhang was filled with biotin-14-dATP (Life Technologies) using Klenow DNA polymerase (New England Biolabs). In contrast to traditional Hi-C,  the ligation was performed when the nuclear membrane was still intact (in situ protocol). DNA was ligated for 4 hours at 16°C using T4 ligase (New England Biolabs). Protein was degraded by proteinase K (New England Biolabs) treatment at 55°C for 30 min. The crosslinking was reversed with addition of 500 mM NaCl and incubation at 65°C overnight. DNA was purified by ethanol precipitation, sonicated to 300-700 bp fragments, and size-selected using SPRI magnetic beads as described 33. Biotinylated DNA was selected with Dynabeads MyOne T1 Streptavidin beads (Life Technologies). Sequencing libraries were prepared on beads, checked using an Agilent Bioanalyzer 2100 and quantified using a Qubit (Life Technologies).
attributes.tag: GEO Accession
attributes.value: GSM3476504