title: GSM3389243: CRE111_E14_BR1; Mus musculus; OTHER
library_construction_protocol: In summary, 10 million E14 cells and MEF were lysed with cross-linked by 1% formaldehyde for 10 min at room temperature. 4C library were prepared using previously published protocol(Stadhouders et al., 2013). In summary, 10 million E14 cells and MEF were lysed with cross-linked by 1% formaldehyde for 10 min at room temperature. Cross-linked chromatin was digested with the primary restriction enzyme HindIII (CRE18, CRE111, CRE132, Oct4 DE; NEB), EcoRI(CRE 140; NEB); Next, 3C library was generated by proximity ligation using T4 DNA ligase (EL0013, Thermo Scientific) on HindIII digested DNA, followed by cross-link removal using Proteinase K (AM2546, Vivantis). The 3C libraries were then subjected to a second restriction enzyme digestion using DpnII (CRE132, Oct4 DE; NEB), NlaIII(CRE18; NEB), CviQI(CRE111, CRE140; NEB) followed by another ligation reaction using T4 DNA ligase. For each viewpoint, 3.2 μg of the resulting 4C templates was used to perform an inverse PCR. The PCR products were then run on 2% agarose gels. On the gel, smears from 200 to 600 bp were excised and unwanted PCR product bands were removed. DNA was then extracted from the cut-out gel pieces for next-generation sequencing on an Illumina Miseq. Interaction regions for each bait sequence was identified using r3Cseq package(Thongjuea et al., 2013).
attributes.tag: GEO Accession
attributes.value: GSM3389243