GSM4065890: BPSM_dBvgA_a-BvgA_CHIPSEQ-RUN2_sample8; Bordetella pertussis; ChIP-Seq
The bacterial suspension was centrifuged at 5,000 x g for 10 min and washed twice with PBS. Cell pellets were resuspended in Immunoprecipitation buffer + Antiprotease. Bacteria were lysed by sonication with a refrigerated Diagenode Bioruptor at 4°C with specific TPX Diagenode tubes. Cell debris were removed by centrifugation (15 min, 4,000 rpm, 4ºC). IP was performed with anti-BvgA antibodies that recognize phosphorylated and non-phosphorylated BvgA and the samples were incubated at 4°C on a wheel for 16h. Protein G Magnetic Dynabeads were added to the IP samples and incubated for 4h at 4°C on the wheel. Beads were separated from the lysate with a magnet and washed with IP buffer. Beads were finally resuspended in TE buffer and incubated for 6h at 65ºC. The surpernatant was then treated with RNAseA and Proteinase K and the DNA fragments were extracted with Phenol-Choloform and precipitated with isopropanol. The DNA fragments isolated by ChIP were used to build the Illumina library using the Illumina TruSeq ChIP Library Preparation Kit, followed by sequencing on an Illumina NextSeq 500 benchtop sequencer on SR150 high output run mode.
GEO Accession
GSM4065890