Illumina HiSeq 2000 sequencing; The transcriptional regulator TFB-RF1 activates transcription of a putative ABC transporter in Pyrococcus furiosus
The transcriptional regulator TFB-RF1 activates transcription of a putative ABC transporter in Pyrococcus furiosus
Cells were grown under anaerobic conditions in nutrient rich medium based on SME in a 15L bio-fermentor and supplemented with 40 mM pyruvate and 0.025 % yeast extract. Cultivation was done 95°C. After the cells reached a cell density of 1 to 1.5x108 cells/ml (middle to late exponential phase) fixation was done directly in the bio-fermenter at 95°C with a final concentration of 0.1% (v/v) formaldehyde. After 20s the crosslinking reaction was stopped by adding glycine to a final concentration of 15 mM and the bio-fermenter was straight away cooled down to 20°C for harvesting the crosslinked P. furiosus cells. Formaldehyde-treated cells were disrupted by sonication using the Branson Sonifier (Branson, Danbury, USA) until an average fragment length of 250 bp to 500 bp was obtained. Insoluble particles were removed by centrifugation. After freezing with liquid nitrogen the cell extracts were stored at -80°C. For determination of DNA concentration and fragment length 1 volume of cell extract was mixed with 4 volumes ChIP elution buffer (10 mM Tris pH 8.0, 1% (w/v) SDS, 0.1 mM EGTA) and incubated overnight at 65°C. After RNase treatment DNA was purified via the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and concentration was measured using the NanoDrop (Peqlab, Erlangen, Germany). For immunoprecipitation (IP) 10 µg of purified polyclonal antibodies raised against TFB-RF11 was coupled to 50 µl Dynabeads Protein G for Immunoprecipitation (Thermo Fisher Scientific, Waltham, USA) according to the manufactures instructions. Antibody coupled magnetic beads were resuspended in 500 µl P. furiosus cell extracts adjusted to a total DNA amount of 15 µg in PBS and incubated overnight at 4°C. Immunoprecipated complexes were washed in total five times with 500 µl of the following washing buffers: 2x low salt buffer, 1x high salt buffer, 1x LiCl detergent and 1 x TE. Elution from the beads was done in 100 µl ChIP elution buffer at 65°C for 10 minutes. A second elution step was done without heating using 150 µl TE buffer supplemented with 0.67% (v/v) SDS and both eluates were combined. For the input sample 200 µl TE supplemented with 1% (v/v) SDS was added to 50 µl not immunoprecipitated P. furiosus cell extract (1,5 µg total DNA, 10% of the IP). Eluted complexes and input samples were incubated overnight at 65°C for reversal of the crosslink. After treatment with RNase A and Proteinase K ChIP enriched and input DNA was purified via the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and stored at -20°C. Library preparations were done according to the NEBNext ®-ChIP-Seq library prep reagent set for Illumina protocol (New England Biolabs, Ipswich, USA). For multiplex sample preparation the NEBNext® Multiplex oligos (primer set 1 and 2) (New England Biolabs, Ipswich, USA) were used and libraries were PCR amplified by the NEBNext®High Fidelity Master Mix (New England Biolabs, Ipswich, USA).
Experimental Factor: immunoprecipitate
TFB-RF1; PF1088