Puc118 hinc 2 bap vector

The immunoprecipitated DNA was cloned as described previously [22 (link)]. Briefly, the DNA isolated from ChIP was heated at 68°C for 5 min and then cooled to 37°C. One to two units of T4 DNA polymerase were added to the DNA and reaction mix containing repair buffer (18 mM ammonium sulfate, 66 mM Tris [pH 8.0], 6.6 mM MgCl₂, 50 mM β-mercaptoethanol, and 0.5 mM of each nucleotide) and subsequently incubated at 37°C for 15 min. The reaction was terminated by 1 μl of 0.5 M EDTA for a 50 μl reaction mix. The processed DNA was cloned into pUC118 Hinc II/BAP vector (Takara). Each ligation was transformed into DH-5α competent cells (Clontech). The entire transformation was plated onto ampicillin treated Luria broth agar plates. Randomly picked 120 colonies with inserts were identified by PCR using M13 primers spanning the cloning site in the vector. Inserts > 200 bp were selected for sequencing using a capillary automatic sequencer (3730 DNA Analyzer, Applied Biosystem). The BLAST search of the human genome database at NCBI was performed to locate sequences. The possible genomic binding sequences were identified by pattern matching. Specific sequences were also analyzed using BLAST adjusted to short sequences (Program = blastn, Word size = 7, Expect Value = 100, Filter = disabled). The sequence logo was generated by WebLogo (http://weblogo.berkeley.edu/logo.cgi).

The immunoprecipitated DNA was cloned as described previously (Yoon et al., 2015 ). Briefly, the DNA isolated from ChIP was heated at 68 °C for 5 min and then cooled to 37 °C. One to two units of T4 DNA polymerase were added to the DNA and reaction mix containing repair buffer (18 mM ammonium sulfate, 66 mM Tris [pH 8.0], 6.6 mM MgCl₂, 50 mM β-mercaptoethanol, and 0.5 mM of each nucleotide) and subsequently incubated at 37 °C for 15 min. The reaction was terminated by 1 μl of 0.5 M EDTA for a 50 μl reaction mix. The processed DNA was cloned into pUC118 Hinc II/BAP vector (Takara). Each ligation was transformed into DH-5α competent cells (Clontech). The entire transformation was plated onto ampicillin treated Luria broth agar plates. Randomly picked 120 colonies with inserts were identified by PCR using M13 primers spanning the cloning site in the vector. Inserts > 200 bp were selected for sequencing using a capillary automatic sequencer (3730 DNA Analyzer, Applied Biosystem). The BLAST search of the human genome database at NCBI was performed to locate sequences. The possible genomic binding sequences were identified by pattern matching. Specific sequences were also analyzed using BLAST adjusted to short sequences (Program = blastn, Word size = 7, Expect Value = 100, Filter = disabled). The sequence logo was generated by WebLogo (http://weblogo.berkeley.edu/logo.cgi).

Detail information for the cloning and transformation has been previously published (Kim et al. 2012 (link)). The primers used to amplify the 16S rRNA genes for bacteria were 27F and 1492R (Lane 1991 ). The amplification conditions for the PCR were 95°C for 5 min, followed by 30 cycles of 95°C for 45 sec, 55°C for 45 sec, and 72°C for 90 sec, with a final extension step for 5 min at 72°C. PCR products were purified with a QIAquick PCR purification kit (Qiagen, Valencia, CA) and ligated into a pUC118 HincII/BAP vector (Takara Bio Inc.), which was transformed into competent Escherichia coli DH5α cells (Invitrogen Corp., Carlsbad, CA) using heat shock. Plasmids from E. coli DH5α transformants were isolated using the PureLink Quick Plasmid Miniprep kit (Invitrogen Corp.). The 16S rRNA genes from the bacterial clones were sequenced using an Applied BioSystems model 3730xl automated DNA sequencing system (Foster City, CA).