Project description:This SuperSeries is composed of the following subset Series: GSE27310: Slf1p and Sro9p affinity isolations GSE27311: SLF1 overexpression versus control cells Refer to individual Series
Project description:This SuperSeries is composed of the following subset Series: GSE24002: Gis2 overexpression versus control cells GSE24003: Gis2 affinity isolations GSE24004: Gis2 mutant versus wild-type cells Refer to individual Series
Project description:To identify RNAs specifically associated with Slf1p and Sro9p, cells expressing TAP-tagged Slf1, Sro9 or Fpr1 (negative control) were grown to mid-log phase in synthetic complete medium and harvested by centrifugation. RNA affinity isolations were essentially performed as described (Gerber et al. 2004 PLoS Biol.). In brief, TAP-tagged protein were captured from cell extracts with IgG coupled agarose beads (Sigma) and released by incubation with a site-specific protease (AcTEV, Sigma). from the extract (input) and from the affinity isolates was purified with the RNeasy Mini/ Micro Kit (Qiagen). RNAs were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast oligo microarrays over night at 42 degrees in formamide-based hybridization buffer.
Project description:To identify RNAs specifically associated with potential RBPs, yeast cells expressing TAP-tagged RBP or the wild-type strain BY4741 (mock control) were grown to mid-log phase in rich medium and harvested by centrifugation. RNA affinity isolations were essentially performed as described (Gerber et al. 2004 PLoS Biol.; see protocol). In brief, TAP-tagged protein were captured from cell extracts with IgG coupled agarose beads (Sigma) and released by incubation with a site-specific protease (AcTEV, Sigma). from the extract (input) and from the affinity isolates was purified with the RNeasy Mini/ Micro Kit (Qiagen). RNAs were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast oligo microarrays over night at 42 degrees in formamide-based hybridization buffer. Antigenic peptide used in IP: TAP-tag
Project description:To identify RNAs specifically associated with Gis2p, cells expressing TAP-tagged Gis2 or the wild-type strain BY4741 (mock control) were grown to mid-log phase in rich medium and harvested by centrifugation. RNA affinity isolations were essentially performed as described (Gerber et al. 2004 PLoS Biol.; see associated protocol 526). In brief, TAP-tagged protein were captured from cell extracts with IgG coupled agarose beads (Sigma) and released by incubation with a site-specific protease (AcTEV, Sigma). from the extract (input) and from the affinity isolates was purified with the RNeasy Mini/ Micro Kit (Qiagen). RNAs were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast oligo microarrays over night at 42 degrees in formamide-based hybridization buffer. Set of arrays that are part of repeated experiments Genotype: TAP-tagged Gis2 expressing or wild-type strain BY4741 (wt/gis2)