title: GSM1481233: HiCap_replicate2_mESC; Mus musculus; OTHER
library_construction_protocol: Cells were cross-linked with 1% formaldehyde for 10 minutes, lysed and nuclei were isolated. Isolated nuclei were digested with 4-cutter FastDigest MboI (Thermo Scientific, 1µl/µg DNA) for 4 hours at 37 °C. The ends of digested material were filled with biotinylated dATP, dGTP, dCTP and dTTP using Klenow fragment (Fermentas, 0.1 U per 1 µg DNA).  Klenow was deactivated using 0.01 M EDTA at incubating 75 °C for 15 minutes. Then the material was diluted to 3.5 ng/µl and ligated using T4 DNA Ligase (Promega). The crosslinking was reversed by adding Proteinase K and incubating overnight at 65 °C. The proteins were removed and DNA was purified using phenol-chloroform followed by ethanol precipitation. Biotinylated but unligated ends were removed using T4 DNA polymerase by incubating at 12 °C for 15 minutes. The material was fragmented to 300-600 bases by sonication. The fragment ends were repaired and A-tailed. Then the biotinylated fragments were bound to streptavidin beads and unbound fragments were washed away. Sequencing adapters were then ligated to the fragments bound to beads. The material was amplified 6-9 cycles while bound to beads to obtain sufficient amount for sequence capture. Original biotinylated material was removed, supernatant was hybridized to sequence capture probe set according to manufacturer’s instructions (Roche Nimblegen Inc.). Hybridized material was washed according to manufacturer’s instructions and amplified with PCR for 3-6 cycles. Hybridization of the probes to the Hi-C material was done exactly according to the manufacturer’s instructions (Roche Nimblegen Inc). Libraries were prepared for sequencing using Illumina TruSeq protocol
attributes.tag: GEO Accession
attributes.value: GSM1481233