title: Fnr1-Total
organism: Herbaspirillum seropedicae SmR1
description: Protocols: H. seropedicae SmR1 (wild type), Fnr1-3xFlag (fnr1::[Leu-Glu]-3XFlag) and Fnr3-3xFlag (fnr3::[Leu-Glu]-3XFlag) were grown as described in the growth protocol. Immediately after the oxygen switch, cells were cross linked with 1% (vol/vol) formaldehyde (Sigma#F8775) for 25 min. Cross linking was quenched by incubating cells on ice for 5 minutes with 125 mM of Glycine. Cells were collected by centrifugation and washed twice in 20 mL of PBS buffer pH 7.4 (Sigma#P4417). The washed pellets were then resuspended in 1.0 mL of IP lysis buffer (10mMTris-Cl, pH 8.0, 50 Mm NaCl), containing protease cocktail inhibitor (Roche#11836170001). Cell lysis and combined DNA shearing were performed by sonication. Herbaspirillum seropedicae strains were grown at 30°C in NFbHP-Malate medium supplemented with 20 mM of NH4Cl upon switch from 350 rpm to 120 rpm (low oxygen) After cell lysis and DNA shearing, the obtained lysate was clarified by centrifugation at 4°C, 17000 x g for 5 min and 25 μL of the supernatant combined with 75 μL of TE buffer (10 mM Tris-Cl, pH 7.4, 1 mM EDTA), was treated with 1 μL of RNase (Sigma#R4642) and extracted once with phenol:chlorofrom:isoamyl alcohol (25:24:1) followed by chloroform extraction. Verification of DNA shearing was confirmed by loading 10 μL of purified DNA onto a 1% agarose gel. Fragments ranged from 100 to 500 bp, centered on 300 bp. Subsequently, 525 μL of IP Buffer (50 mM Tris-Cl, pH 8.0, 250 Mm NaCl, 0.8% [vol/vol] Triton X-100), containing protease cocktail inhibitor, was added, to the remainder of the lysate (975 μL) and samples were chilled on ice. 50 μL of each lysate was set aside for total-DNA extraction. The remaining lysates were added to 45 μL of EZview Red ANTI-FLAG M2 affinity gel (Sigma#F2426) prepared and washed with TBS buffer (Sigma#T5030) as described in the manufacturer's instructions. The cross-linked lysates were immuno precipitated by incubation for 20 hours on a rotating wheel at 4°C. The samples were then centrifuged for 1 min at 4°C at 4,500 X g, and the pellets were washed once with 1.5 mL of IP Buffer and then three times more with 1 mL of IP buffer. After washing, the affinity gel pellets and 50 μL of total cell extracts (set aside earlier) were incubated overnight at 65°C in 100 μL of IP elution buffer (50mMTris-Cl, pH 7.6, 10mMEDTA, 1% SDS). Samples were then centrifuged at 16,000Xg for 5 min to remove the affinity gel. The eluted DNA was transferred to a new tube and the affinity gel was re-extracted with 50 μL of TE buffer followed by a further 5 min incubation at 65°C. Both total DNA and IP DNA samples were adjusted to 200 μLwith TE buffer and incubated with 3 μL of 10 mg/mL of proteinase K (Roche# 03115879001) for 2 h at 55°C. The samples were extracted once with phenol : chloroform : isoamyl alcohol (25:24:1) and once with chloroform and further purified using Macherey-Nagel NucleoSpin® columns (Catalog#740609.50). DNA was eluted in 50 μL of nuclease-free H2O and quantified by using the Nanodrop (Thermo Scientific). Illumina TruSeq ChIP Library Preparation Kit was used to prepare the libraries which were then size-selected to 200 to 300 base pairs.
attributes.tag: ENA first public
attributes.value: 2018-02-26
attributes.tag: ENA last update
attributes.value: 2018-01-18
attributes.tag: External Id
attributes.value: SAMEA104483434
attributes.tag: INSDC center alias
attributes.value: Postdoctoral Researcher
attributes.tag: INSDC center name
attributes.value: Postdoctoral Researcher
attributes.tag: INSDC first public
attributes.value: 2018-02-26T17:02:53Z
attributes.tag: INSDC last update
attributes.value: 2018-01-18T11:33:50Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6395:Fnr1-Total
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: Fnr1-3xFlag
attributes.tag: sample name
attributes.value: E-MTAB-6395:Fnr1-Total
attributes.tag: strain
attributes.value: SmR1