GSM1567051: anti-FLAG sample; Vibrio cholerae; ChIP-Seq
The ChIP protocol was carried out as described before (Wang H., et al. 2012. Appl Environ. Microbiol. 78(7):2482-8) with modifications. Briefly, nine 100 µl cell lysate samples were combined to perform the immunoprecipitation. The final purified IP DNA was resuspended in 30 µl for a concentration factor of 60 compare to our regular ChIP procedure. The DNA concentration of the anti-FLAG (7.2 ng/µl) and input (23.0 ng/µl) samples were determined by the Qubit® Fluorometric Quantitation System (Life Technologies). Twenty ng of anti-FLAG and input DNA samples were used for library preparation for the Illumina sequencing using the TruSeq ChIP kit (Illumina, San Diego, CA) following manufacturer’s protocol. Briefly, DNA samples were end-repaired and phosphorylated using a combination of Klenow, T4 DNA Polymerase and T4 polynucleotide kinase.  An A base was added to the newly blunt ended molecules followed by adaptor ligation is a standard ligation reaction. Utilizing adaptors than contain a 6 bp index it was run several samples per lane in the HiSeq2000 instrument (Illumina). The ChIP-Seq libraries were then amplified using PCR, quantitated with qPCR and loaded to a standard concentration of 2.5 pM per lane. A standard paired-end sequencing reaction was done generating 50 bp of sequence from each direction of sequencing. The raw data was converted from .bcl file format to .fastq file format for downstream analysis. This was done using CASAVA v1.8.2 software from Illumina.