title: ChIP Seq of HU-GFP in S. pneumoniae; Replicate 1
design: Cells were grown in 2 mL of C+Y, 37C up to OD600 ~ 0.4, diluted 50 times in 30 mL of preheated C+Y (37C) and allowed to grow up to an OD600 of 0.2. The cells were harvested and fixated in 1% formaldehyde. The cells were washed several times in ice-cold PBS and the pellets were snap-frozen and stored in -80C. Subsequently, the cells were thawed and sheared in a Covaris S2 sonicator using the following protocol: 200 cycles, 100 W, 10% load, 12 min. A fraction of the cell mixture was taken apart (input fraction). Polyclonal anti-GFP (ThermoFisher A-6455) was bound to magnetic beads (Dynabeads Protein G 10004D). The remaining sheared cells (IP fraction) were added to the beads and the mixture was incubated for two hours on a rotating wheel for two hours. After immunoprecipitation, the beads were washed. Cross-linking was reversed in both the input and IP fraction by shaking 1400 rpm overnight at 65C in 1% SCS, 10 mM EDTA and 50 mM Tris (pH 8). DNA was purified using phenol-chloroform extraction followed by a PCR purification kit (Qiagen QIAquick). Libraries were prepared by the Lausanne Genomic Technologies Facility (LGTF) using an Ovation Ultralow V2-DNA-Seq library preparation kit (NuGEN) and 100 nt paired-end sequencing was done at the LGTF on an Illumina HiSeq.
identifiers.namespace: SUB14752130
identifiers.id: HUGFP_R1