GSM8283860: HMTS detect chromosomal translocation in HCT116 cell  NCAPH2-AID cell without Auxin treated induced DSB by ETO, rep3; Homo sapiens; OTHER
Genome DNA extract with cell lysis buffer (200 mM NaCl, 10 mM Tris-HCl (pH 7.4), 2 mM EDTA (pH 8.0) and 0.2% (wt/vol) SDS) with  200 µg/ml Proteinase K, and then added equal valume of isopropanol to precipitate DNA. Wahsed three time by 75% EtOH. Air dry and then dissolved in ddH2O Resuspend 1 × 107mammalian cells (previous lentivirus-mediated generation of baits and prey DSBs) in 500 μl of cell lysis buffer (200 mM NaCl, 10 mM Tris-HCl (pH 7.4), 2 mM EDTA (pH 8.0) and 0.2% (wt/vol) SDS) with 200 ug/ml Proteinase K and incubate them at 56 °C overnight (16 h). Add 500 μl isopropanol directly into the microtube and mix immediately by inverting the microtube until the genomic DNA can be seen to form a pellet. Using a tip, transfer the DNA pellet to a new microtube containing 1 ml 75% ethanol. Repeat two times and then use a pipette remove the 75% ethanol, air dry the DNA pellet at RT. Check the concentration of a 1-μl aliquot with a NanoDrop; theA260/280value should be above 1.8. Take 1 ul to run gel to check the integration of the genome DNA. Then the DNA cleavage by MluCI Restrict enzyme. After cleavage, Run 1% Agrose gel to purify DNA fragment >1.0 kb (bait region >1.2 kb) with Thermo  Fisher gel Extraction Kit. Next, self-ligation by T4 DNA ligase in low DNA concentration (1.6ng/ul). After ligation, use I-SceI or I-CeuI cut to remove possible non-translocation fragments. Subsequently, perform 1 st round PCR with the ISPCR primer and then purified with QIAquick PCR Purification Kit. Then perform 2nd PCR with Indexed Illuminal sequencing primer. And purified with  QIAquick PCR Purification Kit, measure DNA concentration with Qubit and then do Illumina pair-end 300 bp Mi-seq with regular illumine sequencing primer or custom primer.