Project description:We developed a strategy to map GlcNAc modified proteins based on a chemical chromatin precipitation strategy. Drosophila S2 cells, as well as wild type (wt) and sxc null pupae, were fed with 100 uM Ac4GalNAz and the resulting DNA was cross-linked, sonicated, and purified. DNA strands cross-linked to GlcNAz proteins were isolated by congugating with alkyn-biotin by Staudinger ligation followed by streptavidin purification. The resulting DNA was constructed into libraries for sequencing. To asses the robustness of our strategy, we compared GalNAz ChIP-seq results in S2 cells with two other GlcNAc ChIP-seq strategies, using a mutant β-1,4-galactosyltransferase (GalT) and the lecting wheat germ agglutinin (WGA). Briefly, GalT was incubated with cross-linked, sonicated and purified DNA along with UDP-GalNAz. Ligation to biotin with click chemistry followed by streptavidin purification resulted in library ready material. For WGA and Pho ChIP-seq, cross-linked and purified DNA was incubated with pho antibody or WGA resin and purified followed by library preperation.
Project description:RNA co-immunoprecipitations with C-terminal protein A-tagged She proteins. One liter of cells were cultured at 30 degrees in YPAD medium and collected during exponential growth by centrifugation. Cells were washed twice and broken mechanically with glass beads. Extracts were incubated with IgG-agarose beads (Sigma). The beads were washed four times, and She proteins were released from the beads by cleavage with TEV-protease (Invitrogen). RNA was isolated by phenol/chloroform extraction and isopropanol precipitation from TEV eluates, which corresponds to the purified fraction, and from extracts (input). Both RNA samples, input and purified, were reverse transcribed and labeled with the fluorescent dyes Cy3 and Cy5 (Amersham), respectively. The samples were mixed and competitively hybridized to yeast DNA microarrays containing all yeast genes
Project description:RNA co-immunoprecipitations with C-terminal protein A-tagged She proteins. One liter of cells were cultured at 30 degrees in YPAD medium and collected during exponential growth by centrifugation. Cells were washed twice and broken mechanically with glass beads. Extracts were incubated with IgG-agarose beads (Sigma). The beads were washed four times, and She proteins were released from the beads by cleavage with TEV-protease (Invitrogen). RNA was isolated by phenol/chloroform extraction and isopropanol precipitation from TEV eluates, which corresponds to the purified fraction, and from extracts (input). Both RNA samples, input and purified, were reverse transcribed and labeled with the fluorescent dyes Cy3 and Cy5 (Amersham), respectively. The samples were mixed and competitively hybridized to yeast DNA microarrays containing all yeast genes Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
Project description:RNA co-immunoprecipitations with C-terminal protein A-tagged She proteins. One liter of cells were cultured at 30 degrees in YPAD medium and collected during exponential growth by centrifugation. Cells were washed twice and broken mechanically with glass beads. Extracts were incubated with IgG-agarose beads (Sigma). The beads were washed four times, and She proteins were released from the beads by cleavage with TEV-protease (Invitrogen). RNA was isolated by phenol/chloroform extraction and isopropanol precipitation from TEV eluates, which corresponds to the purified fraction, and from extracts (input). Both RNA samples, input and purified, were reverse transcribed and labeled with the fluorescent dyes Cy3 and Cy5 (Amersham), respectively. The samples were mixed and competitively hybridized to yeast DNA microarrays containing all yeast genes Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
Project description:Characterisation IER3-AS1 interacting proteins using chromatin oligo-affinity precipitation (ChOP) followed by mass spectrometry. The HeLa cell lysates was incubated with biotinylated antisense oligonucleotides (ASO), targeting an experimental target antisense long noncoding RNA IER3-AS1 or a control RNA LacZ. LacZ and IER3-AS1 interacting proteomes were pulldown using Streptavidin beads. The eluted protein samples from both LacZ control ASOs and IER3-AS1 ASOs subjected to mass-spectrometry analyses to identify IER3-AS1 interacting proteins.