Homology-mediated End-capping as a primary step of Sister Chromatid Fusion in the Breakage-Fusion-Bridge cycles
A colorectal cancer cell line Colo320DM and primary fibroblast IMR90 were obtained from ATCC. Genomic DNA was processed for GAPF (Genome Wide Association of Palindrome Formation) as previously described with minor modifications. A total of two μg of genomic DNA was used as starting material, of which 1 μg was digested with KpnI and 1 μg was digested with SbfI for 16 hours (20 μL reaction with 10 U of each enzyme). After  the heat inactivation of the enzyme (65°C for 20 min), digests were combined for denaturation (7 minutes in boiling water) and rapid renaturation in the presence of 100mM NaCl and 50% formamide (90 μL reaction mix consisting of 40 μL of DNA digests, 3 μL of 3M NaCl, 45 μL of formamide and 2 μL of water).  Following the digestion with nuclease S1(Invitrogen) (120 μL reaction consisting of 90 μL of DNA, 8 μL of 3M NaCl, 12 μL of S1 nuclease buffer, 2 μL of 100U/μL S1 nuclease and 8 μL of water), DNA was purified using the ChargeSwitch PCR Clean-up Kit.  After the fragmentation using M220 focused ultrasonicator (Covaris), fragments between 100 –250 bp were purified for NGS library construction using 5500 SOLiD™ Fragment Library Core Kit (Applied Biosystems). Each DNA sample was ligated to linkers with unique barcodes. Libraries were then pooled together and sequenced in a single flowcell run using SOLiD 5500xl sequencer. The barcodes were then used to trace the sequence data back to a specific sample. BAM files were generated using LifeTech LifeScope software. Read depth analyses were done using Genomics Suite (Pertek). All repeat content analysis was performed using the open, web based platform GALAXY and FASTQ files. Briefly, Bowtie was used to map the reads in hg19.  We used the commonly used settings except for allowing for a 3-bp mismatch penalty for the 75-bp reads.  The resulting SAM files were converted to BAM files using SAM Tools.