DNA from FLAG-RocS pulldown, replicate 2
The protocol for immunoprecipitation of FLAG-tagged RocS was largely based on Minnen et al., Mol. Micro, 2011 and was performed in duplicate. Specifically, cells were pre-cultured in acid C+Y (pH 6.8) and grown until OD600 0.40. Cells were then diluted 1:50 in acid C+Y, to a final volume of 30 mL and grown until OD600 0.20. Fixation, formaldehyde quenching and harvesting was performed as described earlier. DynabeadsTM Protein G (Invitrogen) were prepared according to the manufacturers instructions and loaded with 10 ug of anti-FLAG antibody. Cell pellets were resuspended in 2 mL ice-cold lysis buffer and transferred to a 5 ml round-bottom tube. Samples were sonicated on ice for 5-10x 30 sec  on a Sonics Vibracell VCX130 with 65% amplitude. Samples were then split into 200 L whole-cell extract (WCE, stored at -20C) and 800 L for immunoprecipitation. Immunoprecipitation was performed as described before. WCE samples were thawed and combined with 300 L TES buffer and 20 L of 10% SDS. To elute DNA, both WCEs and immunoprecipitates (IPs) were incubated overnight on a shaker at 65C. On a magnet, the DNA-containing supernatant was transferred to a fresh tube.  DNA was purified by phenol-chloroform extraction, followed by ethanol precipitation.Further library preparation was performed by GATC Biotech. Due to an insufficient amount of material in one of the immunoprecipitation samples, we collected data on 2 WCE samples and 1 IP sample.