GSM7947483: WT_T4_nobrg1i_ctrl_5; Homo sapiens; OTHER
Cells were harvested and cross-linked with 2% formaldehyde for 10 min at room temperature, lysed and digested with MboI (New England Biolabs, Cat# R0147L) for three rounds of 4h each. Then first ligation step was performed by T4 DNA ligase (New England Biolabs, Cat# M0202L), and ligated DNA was decrosslinked and purified. A second digestion step was performed with NlaIII overnight (New England Biolabs, Cat# R0125L). Afterwards, a second ligation step was performed. DNA was purified and measured by Qubit. 5ng of DNA were amplified in PCR using the viewpoint primers. Three viewpoints were used: one close to HR-prone DSB (DSB2), one close to NHEJ-prone DSB (DSB3), and one with no adjacent DSB (ctrl). The PCR products were purified by AMPure XP beads and measured by Qubit. For 4C–seq library preparation, a final qPCR using 2ng DNA was performed with the inverse primers including the Illumina adaptor sequences and a unique index for each condition. DNA libraries were purified by AMPure XP beads and measured by Qubit. Fragment size was checked by Tape station. Libraries were pooled and subjected to 75-bp paired-end sequencing on a Nextseq550 platform at DKFZ Next Generation Sequencing Core Facility.