title: GSM4101228: NbBsrDI-nickase-NT; Escherichia coli str. K-12 substr. DH10B; OTHER
library_construction_protocol: R1:Nick translation of genomic DNA. After creation or conversion of the interested sites into nick sites, split the DNA samples equally into two tubes: one for nick translation and the other for negative control. The nick translation was performed in a 50 μl reaction system containing 2 μl of alpha-thio-dNTPs (2.5 mM each, TRILINK), 1X of NEBcutsmart buffer, 2 μl of DNA polymerase I and DNA template. For the negative control, used H2O instead of DNA polymerase I. The reaction mixture was incubated at 15 °C for 90 min and then terminated by heating at 75 °C for 20 min. The product was ready of template DNA digestion after the purification by DyeEx.  Genomic DNA template digestion. The DNA product from nick translation was digested by Exonuclease III (NEB) and RecJf (NEB) to remove the template DNA. A 50 μl reaction system containing 200 units Exonuclease III, 5 μl NEBcutsmart buffer and DNA product from nick translation was incubated at 37 °C for 60 min to digest double-stranded DNA template. Incubate the reaction at 95 °C for 3 min and then put on ice immediately to denature DNA. And then add 60 units of RecJf, incubate at 37 °C for 60 min. For some DNA samples with high complexity of structure and/or modifications, additional digestion with 60 units of RecJf might be necessary. After digestion, inactivate the enzymes by incubation at 80 °C for 10 min. The product was then purified by Zymo Oligo clean & concentrator kit (Zymo) following the manufacturer's protocol. The purified product was ready for Illumina library preparation.   Nick translation of genomic DNA. After creation or conversion of the interested sites into nick sites, split the DNA samples equally into two tubes: one for nick translation and the other for negative control. The nick translation was performed in a 50 μl reaction system containing 2 μl of alpha-thio-dNTPs (2.5 mM each, TRILINK), 1X of NEBcutsmart buffer, 2 μl of DNA polymerase I and DNA template. For the negative control, used H2O instead of DNA polymerase I. The reaction mixture was incubated at 15 °C for 90 min and then terminated by heating at 75 °C for 20 min. The product was ready of template DNA digestion after the purification by DyeEx.  Genomic DNA template digestion. The DNA product from nick translation was digested by Exonuclease III (NEB) and RecJf (NEB) to remove the template DNA. A 50 μl reaction system containing 200 units Exonuclease III, 5 μl NEBcutsmart buffer and DNA product from nick translation was incubated at 37 °C for 60 min to digest double-stranded DNA template. Incubate the reaction at 95 °C for 3 min and then put on ice immediately to denature DNA. And then add 60 units of RecJf, incubate at 37 °C for 60 min. For some DNA samples with high complexity of structure and/or modifications, additional digestion with 60 units of RecJf might be necessary. After digestion, inactivate the enzymes by incubation at 80 °C for 10 min. The product was then purified by Zymo Oligo clean & concentrator kit (Zymo) following the manufacturer's protocol. The purified product was ready for Illumina library preparation.   R2:Mapping of modification/damage sites on DNA by TdT-dependent method. The steps of DNA fragmentation, blocking and nick conversion were same as described above. 100ng of the nick converted DNA was denatured by heating at 95 °C for 3 min in 20 μl H2O, followed by Illumina library preparation with the Clontech SMART ChIp-seq kit (Clontech) by following the manufacturer's protocol. 12 cycles were used in the final step of PCR amplification. Illumina library preparation. Illumina library preparation was performed by the Clontech SMART ChIp-seq kit (Clontech) by following the manufacturer's protocol. 12 cycles were used in the final step of PCR amplification. The PCR product from each sample was combined with its corresponding negative control and then size selected using AMPure XP beads (NEB). The purified library was submitted to Illumina instrument for paired-end sequencing.
attributes.tag: GEO Accession
attributes.value: GSM4101228