Chromatin Immunoprecipitation of B. burgdorferi

The frozen B. burgdorferi cells were thawed on ice. One hundred microliters of cells were used to prepare DNA samples for whole genome sequencing (see below), and 400 μL were processed for chromatin immunoprecipitation-sequencing (ChIP-seq) as described previously^{99 (link)}. Briefly, the fixed cells were lysed using lysozyme at 4 mg/mL final concentration. Crosslinked chromatin was sheared to an average size of 250 bp by sonication using a Qsonica Q800R2 water bath sonicator. The lysate was precleared using Protein A magnetic beads (Fisher 45-002-511) and was then incubated with 4 μL undiluted anti-GFP antibodies^{100 (link)} or anti-mCherry antibodies^{67 (link)} overnight at 4˚C. The next day, the lysate was incubated with Protein A magnetic beads for 1 h at 4 °C. After washes and elution, the immunoprecipitate was incubated at 65 °C overnight to reverse the crosslinks. The DNA was next treated with RNase A, proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), resuspended in 100 µL of buffer EB (Qiagen), and used for library preparation with the NEBNext UltraII kit (E7645). The library was sequenced using Illumina NextSeq500 at Indiana University’s Center for Genomics and Bioinformatics.