RNA coimmunoprecipitations
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE45794 | GEO | 2013/04/05
SECONDARY ACCESSION(S): PRJNA196328
REPOSITORIES: GEO
Similar Datasets
Project description:Nascent polypeptides emerging from the ribosome during biogenesis can interact with many chaperones and protein homeostasis factors. The high sensitivity of RNA identification can be used to identify substrates of specific cotranslationally acting chaperones. Protein A-tagged (TAP-tag) chaperones associated with translating ribosomes were immunopurified by binding to magnetic IgG beads, washed, and chaperone complexes were eluted with TEV protease treatment. Immunopurified RNAs and total cell extract RNAs were isolated, reverse transcribed, coupled to Cy5 and Cy3 dyes, respectively, and comparatively hybridized to DNA microarrays Set of arrays that are part of repeated experiments
2013-04-05 | E-GEOD-45794 | biostudies-arrayexpress
Project description:In this study, we systematically identified the RNAs associated with a selective sample of 40 of ~600 yeast RNA-binding proteins (RBPs). To identify RNAs associated with each putative RBP, C-terminal tandem affinity purification (TAP)-tagged proteins, expressed under control of their native promoters, were affinity purified from whole cell extracts of cultures grown to mid-log phase in rich medium [1-3]. Extracts were incubated with immunoglobulin G (IgG) agarose beads, washed, and ribonuclear protein complexes were eluted by tobacco etch virus (TEV) protease treatment (Text S2). We performed two to four independent isolations with each tagged strain. As controls, we performed 13 immunoaffinity purifications (IPs) of untagged strains to identify and exclude potential false-positive RNA targets. We purified total RNA from the whole-cell extracts and TEV-purified fractions, reverse transcribed with an amino-allyl-dUTP/dNTP mix, coupled the purified cDNA to Cy3 and Cy5 dyes, respectively, mixed the two differentially labeled cDNA pools, and then hybridized them to DNA microarrays (Datasets S1-S4). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Genotype: strain expressing tagged version of RNA-BP Keywords: all_pairs
2008-10-28 | GSE13135 | GEO
Project description:In this study, we systematically identified the RNAs associated with a selective sample of 40 of ~600 yeast RNA-binding proteins (RBPs). To identify RNAs associated with each putative RBP, C-terminal tandem affinity purification (TAP)-tagged proteins, expressed under control of their native promoters, were affinity purified from whole cell extracts of cultures grown to mid-log phase in rich medium [1-3]. Extracts were incubated with immunoglobulin G (IgG) agarose beads, washed, and ribonuclear protein complexes were eluted by tobacco etch virus (TEV) protease treatment (Text S2). We performed two to four independent isolations with each tagged strain. As controls, we performed 13 immunoaffinity purifications (IPs) of untagged strains to identify and exclude potential false-positive RNA targets. We purified total RNA from the whole-cell extracts and TEV-purified fractions, reverse transcribed with an amino-allyl-dUTP/dNTP mix, coupled the purified cDNA to Cy3 and Cy5 dyes, respectively, mixed the two differentially labeled cDNA pools, and then hybridized them to DNA microarrays (Datasets S1-S4). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Genotype: strain expressing tagged version of RNA-BP Keywords: all_pairs Computed
2010-06-25 | E-GEOD-13135 | biostudies-arrayexpress
Project description:RBP9-TAP was expressed in bloodstream T. brucei and bound mRNA was pulled down using IgG beads and eluted via TEV-protease cleavage. rRNA was depleted using RNAseH and rRNA hybridizing oligos.
2020-05-30 | E-MTAB-9093 | biostudies-arrayexpress
Project description:Procyclic-form trypanosomes (Liser 427) constitutively expressing either TAP-ZC3H20 or TAP-ZC3H21, with no other copy of the relevant gene, were used. The protein was pulled down with an IgG column , then the protein and bound RNA was eluted using TEV protease. RNA was sequenced from unbound (flow-through) and bound (eluate) fractions.
2019-06-02 | E-MTAB-7998 | biostudies-arrayexpress
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).
2009-08-31 | GSE15247 | GEO
Project description:Bloodstream-form trypanosomes (Lister 427) constitutively expressing ZC3H5-TAP (Tb927.3.740) were used. The protein was pulled down with an IgG column , then the protein and bound RNA was eluted using TEV protease. RNA was then sequenced from unbound (flow-through) and bound (eluate) fractions.
2020-06-06 | E-MTAB-9084 | biostudies-arrayexpress
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
2008-08-26 | GSE10279 | GEO
Project description:We aimed to find proteins associated with HNRNPH in bloodstream-form _Trypanosoma brucei_. We integrated a sequence encoding a cleavable TAP (Tandem Affinity Purification) tag into the genome upstream of, and in frame with, one _HNRNPH_ gene. The other allele was deleted. TAP-HNRNPH was purifed from triplicate lysates as shown under "Sample processing protocol". Sample processing protocol (30-5000chracters) The TAP-HNRNPH was bound to IgG beads, then released with Tobacco Etch Virus (TEV) protease. TAP-CFP served as the negative control. The His-tagged TEV protease was then removed using Ni-NTA-magnetic beads. Eluted proteins were separated on a 1.5 mm NuPAGE™ Novex™ 4-12 % Bis-Tris protein gel. For details see doi: 10.1371/journal.pone.0258903
2022-10-15 | PXD037289 | Pride
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
2010-06-20 | E-GEOD-10279 | biostudies-arrayexpress