GSM2366159: eHiC_6; Homo sapiens; OTHER
After confluence, the cells were detached with trypsin and collected by spinning down at 900g for 5 minutes. Then the cells were fixed in 1% formaldehyde for 15 minutes at 37ºC, followed by 1/20 volume of 2.5M glycine at room temperature for 5 minutes to quench formaldehyde. The fixed cells were scrapped, washed in PBS and pelleted before stored in -80C. In easy Hi-C, we begin with the in situ proximity ligation procedure and perform HindIII digestion and proximity ligation while keeping nuclei intact. Lligation was performed directly after HindIII digestion without end repair, leading to HindIII-digestible junction products. After nuclear lysis and reverse crosslink, the DNA are digested with more frequent 4-base cutter DpnII before self-ligation. The samples are next treated with exonuclease to remove DNA that failed to form self-circles, and contaminations from un-ligated ends and other linear DNA species. In order to enrich the ligation junction products, we then cut the self-circularized DNA again with HindIII, and only re-linearized junction DNA will be amplified during the library generation. Libraries were prepared according to Illumina's instruction or cited literatures.