Illumina HiSeq 3000 paired end sequencing; ChIP-Seq to determine the binding of enhancer binding proteins VnfA1 and VnfA3 grown in presence of vanadium or in iron-only conditions in Azotobacter vinelandii DJ.
ChIP-Seq to determine the binding of enhancer binding proteins VnfA1 and VnfA3 grown in presence of vanadium or in iron-only conditions in Azotobacter vinelandii DJ.
For each sample 100 mL of culture was crosslinked with 1% formaldehyde (Sigma #F8775) incubated for 30 min at 30�C with shaking and neutralized with 125 mM of glycine for 5 min on ice. The cells were washed twice in ice-cold PBS pH 7.4 (Sigma #P4417-50TAB) and resuspended in 1 mL of IP lysis Buffer (10 mM Tris-HCl pH 8.0 50 mM NaCl 0.8% Triton X-100 1x protease inhibitor (Roche #11836170001)). A. vinelandii was grown at 30�C and 250 rpm in NIL medium (containing 0.2 g/L MgCl2 90 mg/L CaCl2 0.8 g/L KH2PO4 0.2 g/L K2HPO4 14 mg/L Na2SO4 120 mg/L Fe2(SO4)3 and 2.4 mg/L Na2MoO4) supplemented with 2% sucrose. To examine regulation of alternative nitrogenases in the absence of molybdenum trace Mo (up to 8 ppb) was selectively removed by filtering NIL media through activated charcoal. For with-vanadium conditions vanadium was added at 1uM. Cells were then lysed and the DNA sheared by sonication 15 times at an amplitude of 8 microns to obtain fragments between 200 and 1000 base pairs long. The clarified lysate was diluted to 1/3 to a volume of 1.5 ml with IP buffer (50 mM Tris-HCl pH 8.0 250 mM NaCl 0.8% Triton X-100 1x protease inhibitor) prior immuno-precipitation with EZview Red ANTI-FLAG M2 affinity gel (Sigma#F2426) following the manufacturer instructions. Briefly 45 ?L of beads were washed three times with ice cold TBS (Sigma # T5030-50TAB) and added to the 1500 ?L of cross-linked lysate and incubated in a rotating wheel at 4�C until for 18 hours then washed four times with IP Buffer. The DNA fragments were then eluted from the beads with 100 ?L of IP Elution buffer (50 mM Tris-HCl pH 7.6 10 mM EDTA 1% SDS) overnight at 65�C. The beads were removed by centrifugation and the volume of the supernatant adjusted to 200 ?L with TE buffer (10 mM Tris-HCl pH 7.4 1 mM EDTA) and treated with 3 ?L of proteinase K 10mg/ml (Roche # 03115879001) for 2 hours at 55�C. The samples were cleaned by phenol phenol/chloroform extraction and purified using a Macherey-Nagel NucleoSpin� Gel and PCR Clean-up (Catalog#740609.50) following the manufactured instructions. Library construction was carried out by an external contractor (Genewiz) using the Illumina TruSeq ChIP library preparation kit.