58 | Chapter 3 After reversal of the crosslinking the DNA was purified using phenol-chloroform-isoamylalcohol DNA extraction, the DNA was quantified using the CFX384 (Bio-Rad, Hercules, CA) real-time PCR platform. The PCR master mix contained 5 μl of Sso Fast EvaGreen supermix (Bio-Rad, Hercules, CA) and 0.5 μl of 10 μM reverse and forward primers (Biolegio, Nijmegen, the Netherlands). The final volume was adjusted to 9 μl using PCR-grade water, to which 1 μl of diluted cDNA was added. The following PCR conditions were used: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 65°C for 5 s, and extension at 72°C for 10 s. The fluorescent product was measured by a single acquisition mode at 72°C after each cycle. For distinguishing specific from nonspecific products and primer dimers, a melting curve was obtained after amplification by holding the temperature at 65°C for 15 s followed by a gradual increase in temperature to 95°C at a rate of 2.5°C s−1, with the signal acquisition mode set to continuous. The following primers were used Jk1 forward (5’-TCTGTCAGAGAAGCCCAAGC-3’), Jk1 reverse (GGCTGTACAAAAACCCTCCTC-3’), Jk2 forward 5’-CTGGAGAATGAATGCCAGTG-3’), Jk2 reverse (5’- CCCCCATACAAAAACTGAGC-3’), Jk5 forward (5’- GCTGAGCGAAAACTCGTCTTAAGG-3’) and Jk5 reverse (5’- AAAACCTGCCTGTGAAGCCAGTCC-3’). The binding of NBS1 to the Plac8 region was considered as negative control. Primers that amplify the intronic region of Plac8 on chromosome 5 were used as previously described21. Plac8 was found to be highly active during placental development and embryogenesis22,23. Though random RAG1/2 off-target breaks around this gene cannot be excluded, it is not expected to be a RAG1/2 hotspot as there are no antigen receptor loci located on chromosome 5. To calculate the NBS1 density, we normalized the NBS1 qPCR signal to the H4 qPCR signal at each location assayed and compared it to the NBS1/H4 ratio at Plac8 control. Library preparation and SOLiD Sequencing The massive parallel sequencing was performed on the ABI SOLiD 5500 platform. First, the sonicated ChIP DNA samples were quantified by Quant-iTTM dsDNA High-Sensitivity Assay Kit (Thermo Fisher) according to the manufacturer’s protocol. Next the libraries for high throughput sequencing were prepared using the Fragment Library Preparation kit for 5500 Series SOLiD Systems (Applied Biosystems) according to the manufacturer’s protocol, briefly, the DNA fragments were first blunt-ended and phosphorylated at the 5’end. Next, the DNA fragment size selection was performed on Agencourt AMPure® XP beads and separated on 2% agarose gel. The fragments of 250bp bp size were selected by gel excision. Then, the dA-tail was ligated onto the size-selected DNA using the A-tailing enzyme, and using T4 DNA ligase the P1-T and Barcode-T adaptors and barcodes were ligated onto the DNA fragments. These adaptors/barcodes are designed to be compatible with the reverse-read sequencing on 5500 Series SOLiDTM sequencers. Prior to ligation-mediated PCR, the sample was incubated at 72°C for 20 minutes in PCR mix to let the DNA diffuse
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