title: input_3
organism: Dictyostelium discoideum
description: Protocols: A mixture of Dictyostelium discoideum cells was used, that had been developed from the slug to the culmination stages Cells were fixed with 1% formaldehyde, and DNA was extracted by sonication. The lysate was incubated in RET buffer (50 mM TRIS, pH 8.0, 150 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 2 mM EDTA, 0.05% (v/v) Triton X-100 in 50 mM Tris, pH 8) containing cOmplete EDTA-free protease inhibitor cocktail with anti-GFP antibody and ProteinG-Dynabeads. Beads were washed with RET buffer, TTST buffer (150 mM NaCl, 1% Triton X100, 0.1% SDS, 2 mM EDTA, 0.05% Tween20 in 50 mM Tris-Cl, pH 8) and LiCl buffer (250 mM LiCl, 1% sodium deoxycholate, 1% NP40, 1 mM EDTA in 10 mM TRIS, pH 8), and eluted with 1% SDS and 1 mM EDTA in 50 mM Tris-Cl, pH 7.5. The eluate was reverse cross-linked, treated with RNase and proteinase K, and purified on a QIAquick column. DNA ends were repaired with Klenow polymerase and phosphorylated with T4 polynucleotide kinase. After addition of an A-overhang, pre-annealed adaptors were ligated, and DNA was amplified using Phusion DNA polymerase in the presence of 0.2 M trehalose. Fragments <800 bp were isolated by gel electrophoresis and purified using MinElute columns.
attributes.tag: ENA first public
attributes.value: 2017-12-31
attributes.tag: ENA last update
attributes.value: 2017-08-22
attributes.tag: External Id
attributes.value: SAMEA104223499
attributes.tag: INSDC center alias
attributes.value: School of Life Sciences, University of Dundee
attributes.tag: INSDC center name
attributes.value: School of Life Sciences, University of Dundee
attributes.tag: INSDC first public
attributes.value: 2017-12-31T17:01:45Z
attributes.tag: INSDC last update
attributes.value: 2017-08-22T14:06:40Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6000:input_3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: developmental stage
attributes.value: slug and culmination stages
attributes.tag: genotype
attributes.value: spda-/spdA-YFP
attributes.tag: sample name
attributes.value: E-MTAB-6000:input_3