Puf proteins, RNA co-immunopurification
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE3968 | GEO | 2006/01/04
SECONDARY ACCESSION(S): PRJNA104871
REPOSITORIES: GEO
Similar Datasets
Project description:RNA-coimmunopurifications with TAP-tagged Puf proteins from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control. Cells were grown to midlog phase and harvested by centrifugation. TAP-tagged Puf proteins were affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input)and from purified protein samples by phenol-chloroform extraction. RNA samples 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 DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
2006-01-03 | E-GEOD-3968 | biostudies-arrayexpress
Project description:RNA-coimmunopurifications with TAP-tagged Puf proteins from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control. Cells were grown to midlog phase and harvested by centrifugation. TAP-tagged Puf proteins were affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input)and from purified protein samples by phenol-chloroform extraction. RNA samples 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 DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004.
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Project description:RNA-coimmunopurifications with TAP-tagged Khd1 protein from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control (Gerber et al., 2004). Cells were grown to midlog phase in rich media and harvested by centrifugation. TAP-tagged Khd1 was affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input) and from purified protein samples by phenol-chloroform extraction. 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 DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Strain Name: yeast strain with/without TAP-tagged Khd1 Keywords: all_pairs
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Project description:RNA-coimmunopurifications with TAP-tagged Khd1 protein from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control (Gerber et al., 2004). Cells were grown to midlog phase in rich media and harvested by centrifugation. TAP-tagged Khd1 was affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input) and from purified protein samples by phenol-chloroform extraction. 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 DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Strain Name: yeast strain with/without TAP-tagged Khd1 Keywords: all_pairs Computed
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Project description:RNA co-immunopurification with TAP-tagged Shg1p (a component of the Set1 histone methyltransferase complex, SET1C) from Saccharomyces cerevisiae. An untagged (BY4741) served as a control (Gerber et al. 2004). Cells were grown to mid-log phase in rich media and harvested by centrifugation. TAP-tagged Shg1 was affinity-purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input) and from the purified protein sample with the RNAeasy 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. For the method see experimental procedure for RNA IP (Gerber/Dichtl). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
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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) Biological Replicate
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