GSM3667701: MYO-PT1063 [capture Hi-C]; Homo sapiens; OTHER
Myometrium and leiomyoma tissue samples were cryopulverized into a fine powder and immediately formaldehyde cross-linked with 1% formaldehyde for 10min at room temperature with rotation and then quenched with 0.15M glycine for 10min at room temperature with rotation. In-situ Hi-C protocol was used performed on cross-linked tissue samples with HindIII digestion of chromatin_x000B_for 12-16hrs at 37°C. Labeling of DNA ends was carried out with 28.4μM D-Desthiobiotin-7-dATP and ends were proximity ligated with T4 DNA ligase. Labeled and proximity ligated DNA was isolated by phenol-chloroform extraction and sheared to a size of 300-500bp using a Covaris S2 focused ultrasonicator followed by a 0.5X-0.9X AMPure XP bead size selection. Biotin was removed from unligated ends with T4 DNA polymerase followed by streptavidin bead-based biotin pulldown. DNA was eluted off the beads with 10mM biotin, followed by a 1X AMPure XP bead cleanup. KAPA Hyper Prep kit (Kapa Biosystems, cat # KK8502) was used for end-repair, A-tailing, adapter ligation with TruSeq index adapters, and 12 cycles of PCR amplification, all according to manufacturer's instructions. Previously described custom-designed biotinylated 120-mer RNA baits targeting Ensembl promoters of protein coding and non-coding transcripts were used to capture promoter associated biotinylated and adapter ligated Hi-C DNA from myometrium and leiomyoma samples.