GSM2265434: RedN-Venus IP AsyncRep1; Caulobacter vibrioides NA1000; ChIP-Seq
Immunoprecipitated DNA was extracted as described before (Fumeaux et al., 2014) with the following modifications: PierceTM Protein A/G Magnetic Beads (Thermo Fisher Scientific) and anti-GFP (JL-8) Living Colors® Av Monoclonal Antibody (Clontech Laboratories) were used for protein immunoprecipitation, SDS was excluded from ChIP buffer, Herring sperm DNA was not used to saturate beads and sonication was performed using a Digital Sonifier® S-250D on ice with the 1/8” microtip (Branson Sonic Power Co) with 45% output and 10 cycles of 30 s ON/30 s OFF. Quality and concentration of the ChIP-DNA was assessed by estimating the A260/A280 and A260/A230 ratios with a Nanodrop device (Thermo Scientific).  Library preparation and sequencing was performed by the Yale Center for Genome Analysis as follows: ~10ng of immunoprecipitated DNA was evaluated with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA) using a Quant-iT™ DNA Assay Kit high sensitivity (Thermo Fisher Scientific). ChIP-DNA was further purified using Ampure XP SPRI beads (Beckman Coulter Genomics). The DNA was then end-repaired, A-tailed, adapter ligated and PCR enriched (8 -10 cycles). Indexed libraries that met appropriate cut-offs were quantified by both qRT-PCR and insert size distribution determined with the LabChip GX. Samples with a yield of ≥0.5 ng/ul are used for sequencing. Subsequently, for sample concentrations were normalized to 10 nM and loaded into Illumina Rapid or High-output flow cells (Illumina®, San Diego CA) at a concentration that yields 150-250 million passing filter clusters per lane. Samples were sequenced using 75 bp single or paired-end sequencing on an Illumina HiSeq 2500 according to Illumina protocols. The 6 bp index was read during an additional sequencing read that automatically follows the completion of read 1. A positive control (prepared bacteriophage Phi X library) provided by Illumina was spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time.