Project description:This SuperSeries is composed of the following subset Series: GSE13653: Affinity purification of ribosomes and associated RNAs from stress-treated cells GSE13654: Affinity purification of ribosomes and associated RNAs using tagged Rpl16a and Rpl16b Refer to individual Series
Project description:Translating ribosome affinity purification (TRAP) methods allow cell-specific recovery of polyribosome-associated RNAs by genetic tagging of ribosomes in selected cell populations. In this study, we purified and analysed the polyribosome-associated RNA fraction of zebrafish embryo´s retinas at 22 hours post-fertilization. To this aim, we generated a transgenic strain in which the tagged ribosomes expression is restricted to the retina cells due to the activity of a specific promoter.
Project description:Affinity purification of S. cerevisiae or N. crassa Puf3 from S. cerevisiae cells and identification of associated RNAs by microarray
Project description:In this study, we systematically identified RNAs associated with ribosomes. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a or Rpl16b, expressed under control of thier native promoter, were affinity purified from whole cell extracts of cultures grown to mid-log phase in minimal medium. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. We performed two biological replicates with each protein and analyzed the RNA content using oligo microarrays. Total RNA isolated from extracts of cells expressing Rpl16a-ZZ or Rpl16b-ZZ (input) and from the affinity-purified ribosomes was reverse transcribed with oligo(dT) primers. cDNA was labeled with Cy3 and Cy5 fluorescent dyes, respectively, and competitively hybridized to yeast oligo microarrays. Alternatively, RNA was isolated from sucrose gradient fractions containing 60S subunits, 80S monosomes and polysomes. Here, we performed four biological replicates. Analysis was done with oligo microarrays as described above. A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species. strain_or_line_design
Project description:The distribution of histone variants H2Abbd and macroH2A in 13 regions of the HG18 assembly have been studied using a variant of the ChIP-on-Chip technique. HeLa S3 cell lines expressing tagged histones H2A, H2Abbd or macroHA were obtained using retroviral transfer. DNA fractions associated with tagged histones were isolated using a two-step purification procedure that involved affinity chromatography on a column with anti-FLAG antibodies, followed by affinity chromatography on a Ni-agarose column. The obtained genomic DNA samples were analyzed by hybridization with custom NimbleGene genomic microarrays.
Project description:In this study, we systematically identified RNAs associated with ribosomes. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a or Rpl16b, expressed under control of thier native promoter, were affinity purified from whole cell extracts of cultures grown to mid-log phase in minimal medium. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. We performed two biological replicates with each protein and analyzed the RNA content using oligo microarrays. Total RNA isolated from extracts of cells expressing Rpl16a-ZZ or Rpl16b-ZZ (input) and from the affinity-purified ribosomes was reverse transcribed with oligo(dT) primers. cDNA was labeled with Cy3 and Cy5 fluorescent dyes, respectively, and competitively hybridized to yeast oligo microarrays. Alternatively, RNA was isolated from sucrose gradient fractions containing 60S subunits, 80S monosomes and polysomes. Here, we performed four biological replicates. Analysis was done with oligo microarrays as described above. A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species.
Project description:A tagged ectopic version of the ELAV-like protein Tb927.8.6650 of T. brucei was expressed in stable cell lines and pulled down. Co-purifying transcripts were analyzed by sequencing to identify RNAs associated with Tb927.8.6650. Stable procyclic form cell lines expressing tetracycline-inducible TAP-tagged Tb927.8.6650 were created. Cells were harvested 48h after tet-induction, followed by tandem affinity purification of Tb927.8.6650, extraction of co-purified RNA, and sequencing.
