Illumina HiSeq 3000 paired end sequencing; ChIP-seq of Myxococcus xanthus to identify CdbA DNA binding sites in vivo
ChIP-seq of Myxococcus xanthus to identify CdbA DNA binding sites in vivo
Cultures (50 ml) of each strain were grown to mid-late exponential phase.Crosslinking was initiated by the addition of 1% formaldehyde in the presence of 10 mM sodium phosphate (pH 7.6) for 10 min at room temperature with shaking (100 rpm). After cooling in ice for 30 min, cells were quenched with 125 mM glycine, pelleted, washed twice with phosphate-buffered saline solution and stored at −80°C until further use. Cultures were grown in liquid 1% CTT medium to mid-late exponential phase (OD600 = 0.5–0.9) at 32°C with aeration.  The frozen pellet was thawed and resuspended and lysed in TES buffer (470 μl, 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 100 mM NaCl, 2.2 mg/ml lysozyme) in the presence of 1X complete protease inhibitor cocktail (Roche) for 30 min at 37ºC with shaking (100 rpm). It was mixed with ChIP buffer (550 μl, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8.1, 167 mM NaCl, 1X complete protease inhibitor cocktail), incubated for 10 min at 37°C and sonicated to generate fragments of ∼200-400 bp. The sample was clarified by centrifugation and an aliquot of 50 μl supernatant was kept as the input sample. Samples were mixed with 550 μl ChIP buffer with 0.01% sodium dodecyl sulfate (SDS) and pre-cleaned with 30 μl of protein A magnetic beads (Thermo Scientific™) previously washed with PBS buffer (37 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM K2HPO4) plus 1 mg/ml bovine serum albumin (BSA). 10 μl of monoclonal α-FLAG (Rockland) antibodies was added, incubated at 4°C overnight with rotation and immunoprecipitated (2.5 h, 4°C) with rotation with 30 μl of protein A magnetic Dynabeads previously washed with PBS plus 1 mg/ml BSA. The beads were washed with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.1, 150 mM NaCl), with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.1, 0.5 M NaCl), with LiCl buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris pH 8.1), and twice with TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). The protein–DNA complex eluted from the beads in two 100 μl fractions of elution buffer (1% SDS, 0.1 M NaHCO3) and incubated at 65°C overnight in the presence of 20 µl 3M NaCl to disrupt the crosslinks, treated with 0.5 μl proteinase K (50 mg/ml) at 42°C for 2 h and with 2 µl RNase A (10 mg/ml) at 37°C for 20 min, followed by DNA isolation using PCR product purification kit (Macherey-Nagel). The input sample was also subjected to this cross-link reversal and DNA extraction protocol.  Input DNA was assessed for quality by FEMTOpulse (AATI) and quantified with Quantus (Promega). An Illumina-compatible library was then prepared using the Ovation Ultralow Library kit, NuGEN followed by sequencing on Illumina HiSeq3000 with 2 x 150 bp read mode.