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studies (Keppel et al., 2020; Pfeifer et al., 2016). The strains C. glutamicum ATCC 13032::dtxR-C-linker-His and C. glutamicum ATCC 13032::hrrA-C-twinstrep were cultivated each in triplicates from a fresh agar plate in 20 ml BHI in 100 ml shaking flasks and incubated at 30°C for 8 h in a rotary shaker. Cells were transferred into a second pre-culture with 200 ml CGXII media containing 2%(w/v) glucose for further ~16 h incubation. For the heme condition no iron was added to this pre-culture to starve these cells from iron, while for the iron excess condition the standard amount of iron was used (36 µM FeSO4). From this overnight culture, the main culture was inoculated at an OD600 of 3 in 1 l CGXII with 2% glucose and either 100 µM FeSO4 (Fe excess condition) or 0 µM FeSO4 but 4 µM heme (heme condition) in 5 l shaking flasks. After 2 h cultivation at 30°C in a rotary shaker, cells were harvested (5,000 x g, 4°C, 10 min). The cell pellets were washed once with CGXII without MOPS and then the cells were incubated in 20 ml CGXII without MOPS and 1% formaldehyde for 20 min at RT to cross-link the regulator protein to the DNA. To stop this reaction, incubation with 125 mM glycine for 5 min followed. Then, cells were washed three times with either TNI20 (20 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazol, pH 8) or buffer A (100 mM Tris-HCl, pH 8; importantly w/o EDTA, to avoid disturbing DtxR binding) and the pellets were stored overnight at -80°C. \ No newline at end of file diff --git a/studies/2_ChAP-Seq/protocols/2_CellDisruptionAndPurification.txt b/studies/2_ChAP-Seq/protocols/2_CellDisruptionAndPurification.txt new file mode 100644 index 0000000..6ac7a66 --- /dev/null +++ b/studies/2_ChAP-Seq/protocols/2_CellDisruptionAndPurification.txt @@ -0,0 +1,3 @@ +For cell disruption and purification, the pellets were resuspended in approximately 20 ml TNI20 or buffer A with cOmplete protease inhibitor (Roche, Germany) and 2 mg RNase A (AppliChem, Darmstadt, Germany) and disrupted at 40,000 psi using the Multi Shot Cell Disrupter (I&L Biosystems, Germany). To shear the chromosomal DNA the samples were sonified 2 x 20 s with the Branson Sonifier 250 (Branson Ultrasonics Corporation, CT, USA) and finally supernatant was collected after ultra-centrifugation (150,000 x g, 4°C, 1 h). +The DNA, which was bound by the His-tagged DtxR, was purified using Ni-NTA Agarose column material (Thermo Fisher Scientific, USA) according to manufacturer’s instructions to the gravity flow protocol. Washing of the column was performed using TNI20 and the tagged protein and the bound DNA was eluted with TN buffer with rising imidazole concentrations (20 mM Tris-HCl, 300 mM NaCl, 50/100/200/400 mM Imidazol, pH 8). A Bradford assay was performed to evaluate which eluted fractions will be pooled for further DNA preparation, which were the last three of TNI50 elution and the first three of TNI100. +The DNA bound by the twin-Strep-tagged HrrA was purified using Strep-Tactin XT Superflow column material (IBA Lifesciences, Germany) according to manufacturer’s instructions to the gravity flow protocol. Washing of the column was performed using buffer W (100 mM Tris-HCl, 150 mM NaCl, pH 8) and the tagged protein and the bound DNA was eluted with buffer E (100 mM Tris-HCl, 150 mM NaCl, 15 mM D-biotin, pH 8). diff --git a/studies/2_ChAP-Seq/protocols/3_PurificationOfBoundDNA.txt b/studies/2_ChAP-Seq/protocols/3_PurificationOfBoundDNA.txt new file mode 100644 index 0000000..63d4fdf --- /dev/null +++ b/studies/2_ChAP-Seq/protocols/3_PurificationOfBoundDNA.txt @@ -0,0 +1 @@ +After the purification, 1% (w/v) SDS was added to the eluted (and pooled) fractions and incubated overnight at 65°C. Digestion of the protein was accomplished with the addition of 400 µg/ml Proteinase K (AppliChem GmbH, Germany) at 55°C for 2h. Purification of DNA followed by adding Roti-Phenol/Chloroform/Isoamylalcohol (Carl Roth GmbH, Germany) in a 1:1 ratio to the samples and consequent separation of organic phase using Phase Lock Gel (PLG) tubes (VWR International GmbH, Germany) according to manufacturer’s instructions. The aqueous phase was taken off and mixed with 0.1 of the sample volume of 3 M Na-acetate and double the amount of ice-cold EtOH and incubated for 2 h at -20°C. Centrifugation at 16,000 xg, 4°C, 10 min followed. The DNA was precipitated by adding ice-cold 70 %(v/v) EtOH and after another centrifugation, supernatant was taken off carefully and DNA was dried for 3 h at 50°C and eluted in dH2O. \ No newline at end of file diff --git a/studies/2_ChAP-Seq/resources/.gitkeep b/studies/2_ChAP-Seq/resources/.gitkeep new file mode 100644 index 0000000..e69de29 -- GitLab