GSM3729086: Capture-C-ana.telo-rep3; Mus musculus; OTHER
Capture-C experiments were performed as previously described (Hughes et al. Nat. Genet. 2014, Huang et al. Genes Dev. 2017). 3C library preparation was similar to that of in-situ HiC experiments without biotin nucleotide labeling. Briefly, cells were fixed with 1% PFA and sorted at designated cell cycle stages. DNA was directly ligated after DpnII digestion, extracted with pheno-chloroform and fragmented. Ligated DNA was purified, fragmented and converted into 3C library using NEBNext DNA Library Prep Master Mix Set for Illumina (NEB E6040, M0543L, E7335S). After indexation with NEBNext multiplex oligos, DNA was cleaned up with AMPure XP beads and further amplified with Illumina P5 and P7 primers for 6 cycles. To perform oligo pull-down, 3C libraries were completely dried on a thermal cycler at 60℃. Dried DNA was re-constituted with 13ul hybridization mixture (2.5ul nuclease free water, 7.5ul 2xhybridization buffer, 3ul hybridization component A, Nimblegen SeqCap Hybridization and Wash kit) for 10min at room temperature. DNA was denatured at 95℃ for 10min and then added with 2ul 3pmole biotin-labeled capture probe pool to make a 15ul hybridization system. Hybridization was immediately carried out at 47℃ for more than 24h on a thermal cycler. Hybridized DNA was pulled down with Dynabeads MyOne Streptavidin C1 beads (40ul per capture) and washed with Nimblegen SeqCap Hybridization and Wash kit according to manufacture instruction. Captured DNA was then eluted into 25ul 0.125N NaOH, followed by HCl neutralization. Captured DNA was then purified with AMPure XP beads. Purified DNA was then amplified with Illumina P5 and P7 primer for 12 cycles and PCR products were cleaned up with AMPure XP beads. PCR products were dried on a thermal cycler at 60℃ to initiate a similar second round of capture for further enrichment of targeted DNA. DNA was eluted into 25ul nuclease-free water and subject to sequencing with Illumina NextSeq 500 platform using Illumina sequencing reagents according to manufacturer's instructions.