GSM4395690: TSB807_HPGA_RW_TPCA; Homo sapiens; Hi-C
In-Situ Hi-C was performed as described in Rao et al., 2014 (Rao et al., 2014).  In brief, approximately 1 million BJ fibroblasts (senescent or quiescent via contact inhibition) per experimental condition were collected and treated overnight in MboI restriction enzyme at 37°C rotating at 8 RPM.  Nuclei were then Klenow treated in the presence of biotin-CTP in order to both blunt and biotin label the digested chromatin.  Nuclei were then treated with HC T4 DNA ligase (Enzymatics) to perform in-situ ligation.  Ligated chromatin was then extracted and purified from nuclei via incubation in SDS followed by phenol/chloroform/isoamyl alcohol extraction. Isolated chromatin was then sheared to ~300bp using Covaris sonication and the associated manufacturer's protocol, afterwards fragments >400bp were removed via size selection using AmPure XP bead selection (Beckman-A63880).  Extracted chromatin fragments were then attached to Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific-65601), and were then reblunted via Klenow, followed by poly-A tailing via Exo-Klenow treatment, both reactions on beads.  Illumina TruSeq adapters were ligated to the bead attached chromatin fragments via treatment with HC T4 DNA ligase, after which the library was PCR amplified using NEBNext High Fidelity 2x PCR Master Mix (NEB-M0541S) and Illumina TruSeq amplification primers.  The library was again size-selected via AmPure beads and sequenced using the HiSeq 4000 using 50 cycle paired end runs.